The present invention concerns molecules and a process in which one or more biologically active peptides are incorporated into an Fc domain. In this invention, pharmacologically active compounds may be prepared by a process comprising (a) selecting at least one peptide that modulates the activity of...http://www.google.com.au/patents/US7442778?utm_source=gb-gplus-sharePatent US7442778 - Modified Fc molecules

The present invention concerns molecules and a process in which one or more biologically active peptides are incorporated into an Fc domain. In this invention, pharmacologically active compounds may be prepared by a process comprising (a) selecting at least one peptide that modulates the activity of a protein of interest; and (b) preparing a pharmacologic agent comprising an amino acid sequence of the selected peptide in a loop region of an Fc domain. This process may be employed to modify an Fc domain that is already linked through an N- or C-terminus or sidechain to a peptide or to a polypeptide (e.g., etanercept). This process may also be employed to modify an Fc domain that is part of an antibody (e.g., adalimumab, epratuzumab, infliximab, Herceptin®, and the like). In this way, different molecules can be produced that have additional functionalities, such as a binding domain to a different epitope or an additional binding domain to the precursor molecule's existing epitope. The peptide can be selected, for example, by phage display, E. coli display, ribosome display, RNA-peptide screening, yeast-based screening, chemical-peptide screening, rational design, or protein structural analysis.

Images(22)

Claims(14)

1. A composition of matter of the formula

(X1)a—F1—(X2)b

and multimers thereof, wherein:

F1 is an IgG1 Fc domain comprising SEQ ID NO: 599 modified so that it comprises at least one X3 inserted into or replacing all or part of a sequence selected from SEQ ID NOS: 621, 622, 624, 625, 627, 628, 630, 632, 634, and 636 within a loop region of the IgG1 Fc domain, said loop region being in a non-terminal domain of the Fc domain;

X1 and X2 are each independently selected from -(L1)c-P1, -(L1)c-P1-(L2)d-P2, -(L1)c-P1-(L2)d-P2-(L3)e-P3, and -(L1)c-P1-(L2)d-P2(L3)e-P3-(L4)r-P4;

X3 is independently selected from -(L5)c-P5, -(L5)c-P5-(L6)d-P6, -(L5)c-P5-(L6)d-P6-(L7)e-P7, and -(L5)c-P5-(L6)d-P6-(L7)e-P7-(L8)r-P8;

5. The composition of matter of claim 4, wherein the myostatin binding peptide sequence is selected from SEQ ID NOS: 218 to 509.

6. The composition of matter of claim 4, wherein the EPO-mimetic peptide sequence is selected from SEQ ID NOS: 1 to 27.

7. The composition of matter of claim 4, wherein the TPO-mimetic peptide sequence is selected from SEQ ID NOS: 28 to 99.

8. A modified antibody, comprising an Fc domain, F1, wherein:

F1 is an IgG1 Fc domain comprising SEQ ID NO: 599 modified so that it comprises at least one X3 inserted into or replacing all or part of a sequence selected from SEQ ID NOS: 621, 622, 624, 625, 627, 628, 630, 632, 634, and 636 within a loop region of the IgG1 Fc domain, said loop region being in a non-terminal domain of the Fc domain, wherein:

X3 is independently selected from -(L5)c-P5, -(L5)c-P5-(L6)d-P6, -(L5)c-P5-(L6)d-P6-(L7)e-P7, and -(L5)c-P5-(L6)d-P6-(L7)e-P7-(L8)f-P8;

P5, P6, P7, and P8 are each independently sequences of pharmacologically active peptides;

L5, L6, L7, and L8 are each independently linkers; and

c, d, e, and f are each independently 0 or 1.

9. The modified antibody of claim 8, wherein X3 is inserted into or replaces all or part of a sequence selected from SEQ ID NOS: 623, 626, 629, 631, 633, 635, and 637.

10. The modified antibody of claim 9, wherein X3 is inserted at Leu139/Thr140.

This application claims the benefit of U.S. Provisional Application No. 60/612,680, filed Sep. 24, 2004, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The success of the drug Enbrel® (etanercept) brought to fruition the promise of therapeutic agents modified with the constant domain of an antibody. Antibodies comprise two functionally independent parts, a variable domain known as “Fab”, which binds antigen, and a constant domain known as “Fc”, which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989), Nature 337: 525-31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusion proteins known in the art.

TABLE 1

Fc fusion with therapeutic proteins

Fusion

Therapeutic

Form of Fc

partner

implications

Reference

IgG1

N-terminus

Hodgkin's disease;

U.S. Pat. No. 5,480,981

of CD30-L

anaplastic lymphoma;

T-cell leukemia

Murine Fcγ2a

IL-10

anti-inflammatory;

Zheng et al. (1995), J.

transplant rejection

Immunol. 154: 5590-600

IgG1

TNF

septic shock

Fisher et al. (1996), N. Engl.

receptor

J. Med. 334: 1697-1702; Van

Zee, K. et al. (1996), J.

Immunol. 156: 2221-30

IgG, IgA, IgM, or

TNF

inflammation,

U.S. Pat. No. 5,808,029,

IgE (excluding

receptor

autoimmune disorders

issued Sep. 15, 1998

the first domain)

IgG1

CD4

AIDS

Capon et al. (1989), Nature

receptor

337: 525-31

IgG1,

N-terminus

anti-cancer, antiviral

Harvill et al. (1995),

IgG3

of IL-2

Immunotech. 1: 95-105

IgG1

C-terminus

osteoarthritis;

WO 97/23614, published Jul.

of OPG

bone density

3, 1997

IgG1

N-terminus

anti-obesity

WO 98/28427, filed

of leptin

Dec. 11, 1997

Human Ig Cγ1

CTLA-4

autoimmune disorders

Linsley (1991), J. Exp. Med.

174: 561-9

A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et al. (1995), Science 267: 383-6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only “peptide” length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”).

Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of which is incorporated by reference). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26:401-24.

Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as “E. coli display.” Another biological approach to screening soluble peptide mixtures uses yeast for expression and secretion. See Smith et al. (1993), Mol. Pharmacol. 43: 741-8. Hereinafter, the method of Smith et al. and related methods are referred to as “yeast-based screening.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as “ribosome display.” Other methods employ chemical linkage of peptides to RNA; see, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci. USA 94: 12297-303. Hereinafter, this and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.

In the case of known bioactive peptides, rational design of peptide ligands with favorable therapeutic properties can be completed. In such an approach, one makes stepwise changes to a peptide sequence and determines the effect of the substitution upon bioactivity or a predictive biophysical property of the peptide (e.g., solution structure). Hereinafter, these techniques are collectively referred to as “rational design.” In one such technique, one makes a series of peptides in which one replaces a single residue at a time with alanine. This technique is commonly referred to as an “alanine walk” or an “alanine scan.” When two residues (contiguous or spaced apart) are replaced, it is referred to as a “double alanine walk.” The resultant amino acid substitutions can be used alone or in combination to result in a new peptide entity with favorable therapeutic properties.

Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15: 1266-70. Hereinafter, these and related methods are referred to as “protein structural analysis.” These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.

Conceptually, one may discover peptide mimetics of any protein using phage display and the other methods mentioned above. These methods have been used for epitope mapping, for identification of critical amino acids in protein-protein interactions, and as leads for the discovery of new therapeutic agents. E.g., Cortese et al. (1996), Curr. Opin. Biotech. 7: 616-21. Peptide libraries are now being used most often in immunological studies, such as epitope mapping. Kreeger (1996), The Scientist 10(13): 19-20.

Of particular interest here is use of peptide libraries and other techniques in the discovery of pharmacologically active peptides. A number of such peptides identified in the art are summarized in Table 2. The peptides are described in the listed publications, each of which is hereby incorporated by reference. The pharmacologic activity of the peptides is described, and in many instances is followed by a shorthand term therefore in parentheses. Some of these peptides have been modified (e.g., to form C-terminally cross-linked dimers). Typically, peptide libraries were screened for binding to a receptor for a pharmacologically active protein (e.g., EPO receptor). In at least one instance (CTLA4), the peptide library was screened for binding to a monoclonal antibody.

TABLE 2

Pharmacologically active peptides

Form of

Binding partner/

peptide

protein of interesta

Pharmacologic activity

Reference

intrapeptide

EPO receptor

EPO-mimetic

Wrighton et al. (1996),

disulfide-

Science 273: 458-63; U.S.

bonded

Pat. No. 5,773,569, issued

Jun. 30, 1998 to

Wrighton et al.

C-terminally

EPO receptor

EPO-mimetic

Livnah et al. (1996),

cross-linked

Science 273: 464-71;

dimer

Wrighton et al. (1997),

Nature Biotechnology 15:

1261-5; International

patent application WO

96/40772, published Dec.

19, 1996

linear

EPO receptor

EPO-mimetic

Naranda et al. (1999),

Proc. Natl. Acad. Sci.

USA, 96: 7569-74; WO

99/47151, published

Sep. 23, 1999

linear; C-

c-Mpl

TPO-mimetic

Cwirla et al. (1997)

terminally

Science 276: 1696-9; U.S.

cross-linked

Pat. No. 5,869,451, issued

dimer

Feb. 9, 1999; WO 00/24770,

published May 4, 2000;

U.S. patent application Ser. No.

2003/0176352, published

Sep. 18, 2003; WO

03/031589, published

Apr. 17, 2003

disulfide-

stimulation of

Paukovits et al. (1984),

linked dimer

hematopoiesis

Hoppe-Seylers Z.

(“G-CSF-mimetic”)

Physiol. Chem. 365: 303-11;

Laerum et al. (1988),

Exp. Hemat. 16: 274-80

alkylene-

G-CSF-mimetic

Bhatnagar et al. (1996), J.

linked dimer

Med. Chem. 39: 3814-9;

Cuthbertson et al. (1997),

J. Med. Chem. 40: 2876-82;

King et al. (1991),

Exp. Hematol. 19: 481;

King et al. (1995), Blood

86 (Suppl. 1): 309a

linear

IL-1 receptor

inflammatory and

U.S. Pat. No. 5,608,035;

autoimmune diseases

U.S. Pat. No. 5,786,331;

(“IL-1 antagonist” or

U.S. Pat. No. 5,880,096;

“IL-1ra-mimetic”)

Yanofsky et al. (1996),

Proc. Natl. Acad. Sci. 93:

7381-6; Akeson et al.

(1996), J. Biol. Chem. 271:

30517-23; Wiekzorek et

al. (1997), Pol. J.

Pharmacol. 49: 107-17;

Yanofsky (1996), PNAs,

93: 7381-7386.

linear

Facteur thymique

stimulation of

Inagaki-Ohara et al.

serique (FTS)

lymphocytes

(1996), Cellular

(“FTS-mimetic”)

Immunol. 171: 30-40;

Yoshida (1984), Int. J.

Immunopharmacol,

6: 141-6.

intrapeptide

CTLA4 MAb

CTLA4-mimetic

Fukumoto et al. (1998),

disulfide

Nature Biotech. 16: 267-70

bonded

exocyclic

TNF-α receptor

TNF-α antagonist

Takasaki et al. (1997),

Nature Biotech. 15: 1266-70;

WO 98/53842,

published Dec. 3, 1998

linear

TNF-α receptor

TNF-α antagonist

Chirinos-Rojas ( ), J.

Imm., 5621-5626.

intrapeptide

C3b

inhibition of

Sahu et al. (1996), J.

disulfide

complement activation;

Immunol. 157: 884-91;

bonded

autoimmune diseases

Morikis et al. (1998),

(“C3b-antagonist”)

Protein Sci. 7: 619-27

linear

vinculin

cell adhesion

Adey et al. (1997),

processes-cell growth,

Biochem. J. 324: 523-8

differentiation, wound

healing, tumor

metastasis (“vinculin

binding”)

linear

C4 binding protein

anti-thrombotic

Linse et al. (1997), J. Biol.

(C4BP)

Chem. 272: 14658-65

linear

urokinase receptor

processes associated

Goodson et al. (1994),

with urokinase

Proc. Natl. Acad. Sci. 91:

interaction with its

7129-33; International

receptor (e.g.,

application WO

angiogenesis, tumor

97/35969, published

cell invasion and

Oct. 2, 1997

metastasis); (“UKR

antagonist”)

linear

Mdm2, Hdm2

Inhibition of

Picksley et al. (1994),

inactivation of p53

Oncogene 9: 2523-9;

mediated by Mdm2 or

Bottger et al. (1997) J.

hdm2; anti-tumor

Mol. Biol. 269: 744-56;

(“Mdm/hdm

Bottger et al. (1996),

antagonist”)

Oncogene 13: 2141-7

linear

p21WAF1

anti-tumor by

Ball et al. (1997), Curr.

mimicking the activity

Biol. 7: 71-80

of p21WAF1

linear

farnesyl transferase

anti-cancer by

Gibbs et al. (1994), Cell

preventing activation

77: 175-178

of ras oncogene

linear

Ras effector domain

anti-cancer by

Moodie et al. (1994),

inhibiting biological

Trends Genet 10: 44-48

function of the ras

Rodriguez et al. (1994),

oncogene

Nature 370: 527-532

linear

SH2/SH3 domains

anti-cancer by

Pawson et al (1993),

inhibiting tumor

Curr. Biol. 3: 434-432

growth with activated

Yu et al. (1994), Cell

tyrosine kinases;

76: 933-945; Rickles et al.

treatment of SH3-

(1994), EMBO J. 13: 5598-5604;

mediated disease states

Sparks et al. (1994),

(“SH3 antagonist”)

J. Biol. Chem. 269: 23853-6;

Sparks et al. (1996),

Proc. Natl. Acad. Sci. 93:

1540-4; U.S. Pat. No.

5,886,150, issued Mar.

23, 1999; U.S. Pat. No.

5,888,763, issued Mar.

30, 1999

linear

p16INK4

anti-cancer by

Fåhraeus et al. (1996),

mimicking activity of

Curr. Biol. 6: 84-91

p16; e.g., inhibiting

cyclin D-Cdk complex

(“p16-mimetic”)

linear

Src, Lyn

inhibition of Mast cell

Stauffer et al. (1997),

activation, IgE-related

Biochem. 36: 9388-94

conditions, type I

hypersensitivity

(“Mast cell antagonist”)

linear

Mast cell protease

treatment of

International application

inflammatory disorders

WO 98/33812, published

mediated by release of

Aug. 6, 1998

tryptase-6

(“Mast cell protease

inhibitors”)

linear

HBV core antigen

treatment of HBV viral

Dyson & Muray (1995),

(HBcAg)

infections (“anti-HBV”)

Proc. Natl. Acad. Sci. 92:

2194-8

linear

selectins

neutrophil adhesion;

Martens et al. (1995), J.

inflammatory diseases

Biol. Chem. 270: 21129-36;

(“selectin antagonist”)

European patent

application EP 0 714 912,

published Jun. 5, 1996

linear,

calmodulin

calmodulin antagonist

Pierce et al. (1995),

cyclized

Molec. Diversity 1: 259-65;

Dedman et al. (1993),

J. Biol. Chem. 268: 23025-30;

Adey & Kay (1996),

Gene 169: 133-4

linear,

integrins

tumor-homing;

International

cyclized-

treatment for

applications WO

conditions related to

95/14714, published

integrin-mediated

Jun. 1, 1995; WO

cellular events,

97/08203, published

including platelet

Mar. 6, 1997; WO

aggregation,

98/10795, published

thrombosis, wound

Mar. 19, 1998; WO

healing, osteoporosis,

99/24462, published

tissue repair,

May 20, 1999; Kraft et al.

angiogenesis (e.g., for

(1999), J. Biol. Chem. 274:

treatment of cancer),

1979-1985

and tumor invasion

(“integrin-binding”)

cyclic, linear

fibronectin and

treatment of

WO 98/09985, published

extracellular matrix

inflammatory and

Mar. 12, 1998

components of T

autoimmune conditions

cells and

macrophages

linear

somatostatin and

treatment or

European patent

cortistatin

prevention of hormone-

application 0 911 393,

producing tumors,

published Apr. 28, 1999

acromegaly, giantism,

dementia, gastric ulcer,

tumor growth,

inhibition of hormone

secretion, modulation

of sleep or neural

activity

linear

bacterial

antibiotic; septic shock;

U.S. Pat. No. 5,877,151,

lipopolysaccharide

disorders modulatable

issued Mar. 2, 1999

by CAP37

linear or

pardaxin, mellitin

antipathogenic

WO 97/31019, published

cyclic,

28 Aug. 1997

including D-

amino acids

linear, cyclic

VIP

impotence,

WO 97/40070, published

neurodegenerative

Oct. 30, 1997

disorders

linear

CTLs

cancer

EP 0 770 624, published

May 2, 1997

linear

THF-gamma2

Burnstein (1988),

Biochem., 27: 4066-71.

linear

Amylin

Cooper (1987), Proc.

Natl. Acad. Sci., 84: 8628-32.

linear

Adrenomedullin

Kitamura (1993), BBRC,

192: 553-60.

cyclic, linear

VEGF

anti-angiogenic; cancer,

Fairbrother (1998),

rheumatoid arthritis,

Biochem., 37: 17754-17764.

diabetic retinopathy,

psoriasis (“VEGF

antagonist”)

cyclic

MMP

inflammation and

Koivunen (1999), Nature

autoimmune disorders;

Biotech., 17: 768-774.

tumor growth

(“MMP inhibitor”)

HGH fragment

treatment of obesity

U.S. Pat. No. 5,869,452

Echistatin

inhibition of platelet

Gan (1988), J. Biol.

aggregation

Chem., 263: 19827-32.

linear

SLE autoantibody

SLE

WO 96/30057, published

Oct. 3, 1996

GD1alpha

suppression of tumor

Ishikawa et al. (1998),

metastasis

FEBS Lett. 441 (1): 20-4

antiphospholipid

endothelial cell

Blank et al. (1999), Proc.

beta-2-glycoprotein-

activation,

Natl. Acad. Sci. USA 96:

I (□2GPI)

antiphospholipid

5164-8

antibodies

syndrome (APS),

thromboembolic

phenomena,

thrombocytopenia, and

recurrent fetal loss

linear

T Cell Receptor beta

diabetes

WO 96/11214, published

chain

Apr. 18, 1996.

Antiproliferative,

WO 00/01402, published

antiviral

Jan. 13, 2000.

anti-ischemic, growth

WO 99/62539, published

hormone-liberating

Dec. 9, 1999.

anti-angiogenic

WO 99/61476, published

Dec. 2, 1999.

linear

Apoptosis agonist;

WO 99/38526, published

treatment of T cell-

Aug. 5, 1999.

associated disorders

(e.g., autoimmune

diseases, viral infection,

T cell leukemia, T cell

lymphoma)

linear

MHC class II

treatment of

U.S. Pat. No. 5,880,103,

autoimmune diseases

issued Mar. 9, 1999.

linear

androgen R, p75,

proapoptotic, useful in

WO 99/45944, published

MJD, DCC,

treating cancer

Sep. 16, 1999.

huntingtin

linear

von Willebrand

inhibition of Factor VIII

WO 97/41220, published

Factor; Factor VIII

interaction;

Apr. 29, 1997.

anticoagulants

linear

lentivirus LLP1

antimicrobial

U.S. Pat. No. 5,945,507,

issued Aug. 31, 1999.

linear

Delta-Sleep

sleep disorders

Graf (1986), Peptides

Inducing Peptide

7: 1165.

linear

C-Reactive Protein

inflammation and

Barna (1994), Cancer

(CRP)

cancer

Immunol. Immunother.

38: 38 (1994).

linear

Sperm-Activating

infertility

Suzuki (1992), Comp.

Peptides

Biochem. Physiol.

102B: 679.

linear

angiotensins

hematopoietic factors

Lundergan (1999), J. Periodontal

for hematocytopenic

Res.

conditions from cancer,

34(4): 223-228.

AIDS, etc.

linear

HIV-1 gp41

anti-AIDS

Chan (1998), Cell 93: 681-684.

linear

PKC

inhibition of bone

Moonga (1998), Exp.

resorption

Physiol. 83: 717-725.

linear

defensins (HNP-1, -2,

antimicrobial

Harvig (1994), Methods

-3, -4)

Enz. 236: 160-172.

linear

p185HER2/neu, C-

AHNP-mimetic: anti-

Park (2000), Nat.

erbB-2

tumor

Biotechnol. 18: 194-198.

linear

gp130

IL-6 antagonist

WO 99/60013, published

Nov. 25, 1999.

linear

collagen, other joint,

autoimmune diseases

WO 99/50282, published

cartilage, arthritis-

Oct. 7, 1999.

related proteins

linear

HIV-1 envelope

treatment of

WO 99/51254, published

protein

neurological

Oct. 14, 1999.

degenerative diseases

linear

IL-2

autoimmune disorders

WO 00/04048, published

(e.g., graft rejection,

Jan. 27, 2000; WO

rheumatoid arthritis)

00/11028, published

Mar. 2, 2000.

linear, cyclic

various

inflammatory

U.S. Pat. No. 6,660,843

conditions,

autoimmune disease,

others

linear, cyclic

Ang-2

inhibition of

U.S. patent application Ser. No.

angiogenesis (e.g., for

2003/0229023, published

treatment of tumor)

Dec. 11, 2003; WO

03/057134, published

Jul. 17, 2003; U.S.

2003/0236193, published

Dec. 25, 2003

NGF

chronic pain, migraine,

WO 04/026329,

asthma, hyperactive

published Apr. 1, 2004

bladder, psoriasis,

cancer, other conditions

linked to NGF

myostatin

U.S. Serial No.

10/742,379, filed Dec. 19,

2003; PCT/US03/40781,

filed Dec. 19, 2003

BAFF/TALL-1

B-cell mediated

U.S. 2003/0195156,

autoimmune diseases

published Oct. 16, 2003;

and cancers (e.g.,

WO 02/092620,

lupus, B-cell

published Nov. 21, 2002

lymphoma)

linear

GLP-1

Diabetes, metabolic

syndrome

aThe protein listed in this column may be bound by the associated peptide (e.g., EPO receptor, IL-1 receptor) or mimicked by the associated peptide. The references listed for each clarify whether the molecule is bound by or mimicked by the peptides.

Peptides identified by peptide library screening were for a long time regarded simply as “leads” in development of therapeutic agents rather than as therapeutic agents themselves. Like other proteins and peptides, they would be rapidly removed in vivo either by renal filtration, cellular clearance mechanisms in the reticuloendothelial system, or proteolytic degradation. Francis (1992), Focus on Growth Factors 3: 4-11. As a result, the art used the identified peptides to validate drug targets or as scaffolds for design of organic compounds that might not have been as easily or as quickly identified through chemical library screening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26:401-24; Kay et al. (1998), Drug Disc. Today 3: 370-8.

A more recent development is fusion of randomly generated peptides with the Fc domain. See U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 to Feige et al. (incorporated by reference in its entirety). Such molecules have come to be known as “peptibodies.” They include one or more peptides linked to the N-terminus, C-terminus, amino acid sidechains, or to more than one of these sites. Peptibody technology enables design of therapeutic agents that incorporate peptides that target one or more ligands or receptors, tumor-homing peptides, membrane-transporting peptides, and the like. Peptibody technology has proven useful in design of a number of such molecules, including linear and disulfide-constrained peptides, “tandem peptide multimers” (i.e., more than one peptide on a single chain of an Fc domain). See, for example, U.S. Pat. No. 6,660,843; U.S. Pat. App. No. 2003/0195156, published Oct. 16, 2003 (corresponding to WO 02/092620, published Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003 (corresponding to WO 03/031589, published Apr. 17, 2003); U.S. Ser. No. 09/422,838, filed Oct. 22, 1999 (corresponding to WO 00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023, published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S. Pat. App. No. 2003/0236193, published Dec. 25, 2003 (corresponding to PCT/US04/010989, filed Apr. 8, 2004); U.S. Ser. No. 10/666,480, filed Sep. 18, 2003 (corresponding to WO 04/026329, published Apr. 1, 2004), each of which is hereby incorporated by reference in its entirety. The art would benefit from further technology enabling such rational design of polypeptide therapeutic agents.

SUMMARY OF THE INVENTION

The present invention concerns a process in which at least one biologically active peptide is incorporated as an internal sequence into an Fc domain. Such an internal sequence may be added by insertion (i.e., between amino acids in the previously existing Fc domain) or by replacement of amino acids in the previously existing Fc domain (i.e., removing amino acids in the previously existing Fc domain and adding peptide amino acids). In the latter case, the number of peptide amino acids added need not correspond to the number of amino acids removed from the previously existing Fc domain; for example, this invention concerns a molecule in which 10 amino acids are removed and 15 amino acids are added. In this invention, pharmacologically active compounds are prepared by a process comprising:

a) selecting at least one peptide that modulates the activity of a protein of interest; and

b) preparing a pharmacologic agent comprising an amino acid sequence of the selected peptide as an internal sequence of an Fc domain.
This process may be employed to modify an Fc domain that is already linked through an N- or C-terminus or sidechain to a polypeptide (e.g., etanercept) or to a peptide (e.g., as described in U.S. Pat. App. Nos. 2003/0195156, 2003/0176352, 2003/0229023, and 2003/0236193; WO 00/24770; WO 04/026329). The process described throughout may also be employed to modify an Fc domain that is part of an antibody (e.g., adalimumab, epratuzumab, infliximab, Herceptin®, and the like). In this way, different molecules can be produced that have additional functionalities, such as a binding domain to a different epitope or an additional binding domain to the precursor molecule's existing epitope. The peptide can be selected, for example, by phage display (which is preferred), E. coli display, ribosome display, RNA-peptide screening, yeast-based screening, chemical-peptide screening, rational design, or protein structural analysis or may be a naturally occurring peptide (e.g. PTH, GLP-1).

The invention further relates to molecules comprising an Fc domain modified to comprise a peptide as an internal sequence (preferably in a loop region) of the Fc domain. Molecules comprising an internal peptide sequence are referred to throughout as “Fc internal peptibodies” or “Fc internal peptide molecules.” These molecules are further described herein below.

The Fc internal peptide molecules may include more than one peptide sequence in tandem in a particular internal region, and they may include further peptides in other internal regions. While the putative loop regions are preferred, insertions in any other non-terminal domains of the Fc are also considered part of this invention. Variants and derivatives of the above compounds (described below) are also encompassed by this invention.

The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins.

The primary use contemplated for Fc internal peptide molecules is as therapeutic or prophylactic agents. A selected peptide may have activity comparable to—or even greater than—the natural ligand mimicked by the peptide. In addition, certain natural ligand-based therapeutic agents might induce antibodies against the patient's own endogenous ligand. In contrast, the unique sequence of the vehicle-linked peptide avoids this pitfall by having little or typically no sequence identity with the natural ligand. Furthermore, the Fc internal peptibodies may have advantages in refolding and purification over N- or C-terminally linked Fc molecules. Further still, Fc internal peptibodies may be more stable in both thermodynamically, due to the stabilization of chimeric domains, and chemically, due to increased resistance to proteolytic degradation from amino- and carboxy-peptidases. Fc internal peptibodies may also exhibit improved pharmacokinetic properties.

Although mostly contemplated as therapeutic agents, compounds of this invention may also be useful in screening for such agents. For example, one could use an Fc internal peptibody (e.g., Fc-loop-SH2 domain peptide) in an assay employing anti-Fc coated plates. Fc internal peptibodies may make insoluble peptides soluble and thus useful in a number of assays.

The compounds of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other mammal) in need thereof. Other related aspects are also included in the instant invention.

Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.

DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C show loop regions of Fc domains that may be modified in accordance with this invention. In these structural representations of the CH2 and CH3 domains of Fc, the loop regions may be considered any part of the model not shown as β-sheet (flat arrows) or α-helix (cylinder).

These structures illustrate the high degree of homology in the secondary and tertiary structural conformations within the Fc domains of different IgG subtypes and between species. The x-ray crystal structure coordinates for these structures can be found in the RCSB Protein Data Bank

FIG. 2A shows a sequence of human IgG1 Fc sequence (SEQ ID NO: 599) used for peptibody fusions with predicted loop sequences in boldface. FIG. 2A shows, in the context of the human IgG1 sequence used for this invention, the Fc loop regions in boldface (SEQ ID NOS: 621, 622, 624, 625, 627, 628, 630, 632, 634, and 636), which are suggested by the structures shown in FIGS. 1A, 1B and 1C. Any, or all of the sites shown in boldface may be suitable for full or partial replacement by or insertion of peptide sequences and are considered part of this invention. Specifically preferred internal sites are underlined (SEQ ID NOS: 623, 626, 629, 631, 633, 635, and 637). One preferred site is SEQ ID NO: 631, between Leu139 and Thr140 in the DELTK (SEQ ID NO: 630) loop. Potential loop sites in other Ig subtypes are understood in the art based on the alignments provided in FIGS. 2B and 2C.

FIGS. 2B and 2C show a sequence alignment of human Fc domains from IgA, IgM and IgG subclasses. FIGS. 2B and 2C show exemplary amino acid sequences (SEQ ID NOS: 600 to 607) of human Fc regions from IgA, IgM and IgG subtypes that may be useful in this invention. Also shown in FIGS. 2B and 2C is a consensus sequence (SEQ ID NO: 608).

FIGS. 2B and 2C also show in boldface the preferred internal sites for peptide addition that correspond to those of the Fc sequence shown in FIG. 2A (SEQ ID NO: 599). In particular, FIGS. 2B and 2C show as such preferred sites the following:

SEQ ID NO: 621 as shown in boldface within SEQ ID NOS: 603 to 608;

SEQ ID NO: 622 within SEQ ID NOS: 603 to 606 and 608;

SEQ ID NO: 638 within SEQ ID NO: 607;

SEQ ID NO: 624 within SEQ ID NO: 603 to 608;

SEQ ID NO: 625 within SEQ ID NOS: 603 and 604;

SEQ ID NO: 639 within SEQ ID NOS: 605 to 608;

SEQ ID NO: 627 within SEQ ID NOS: 603 to 605, 607, and 608;

SEQ ID NO: 640 within SEQ ID NO: 606;

SEQ ID NO: 628 within SEQ ID NOS: 603, 604, and 608;

SEQ ID NO: 641 within SEQ ID NO: 605;

SEQ ID NO: 642 within SEQ ID NO: 606;

SEQ ID NO: 643 within SEQ ID NO: 607;

SEQ ID NO: 630 within SEQ ID NO: 603;

SEQ ID NO: 644 within SEQ ID NOS: 604 to 608;

SEQ ID NO: 632 within SEQ ID NOS: 603, 604, 606, 607, and 608;

SEQ ID NO: 645 within SEQ ID NO: 605;

SEQ ID NO: 634 within SEQ ID NOS: 603, 604, and 607;

SEQ ID NO: 646 within SEQ ID NOS: 605, 606 and 608;

SEQ ID NO: 636 within SEQ ID NOS: 603, 604, 606, and 608;

SEQ ID NO: 614 within SEQ ID NO: 605; and

SEQ ID NO: 620 within SEQ ID NO: 607.

The sequence alignments of FIGS. 2B and 2C suggest two more potential insertion sites at Q167/P168 and/or G183/S184 (using the numbering of SEQ ID NO: 599 in FIG. 2A). These positions correspond to gaps in the IgG sequences where there are 2 and 3 residue insertions found in the aligned IgA and IgM sequences. Other preferred insertion sites correspond to the sequence in FIG. 2A. The preferred insertion sites are underlined FIGS. 2B and 2C and are as follows:

FIG. 3A shows the amino acid sequence (SEQ ID NO: 612) of a human IgG1 Fc domain having insertion of a myostatin binding peptide (SEQ ID NO: 365). Hereinafter, this molecule is referred to as the “myostatin loop peptibody” or “Fc-loop-myo7.” The inserted peptide is shown in boldface and the glycine linkers in italics.

FIG. 3B shows the amino acid sequence (SEQ ID NO: 613) of a C-terminally linked peptibody referred to as TN8-19-07. This peptibody incorporates the same peptide sequence as Fc-loop-myo7 (SEQ ID NO: 365). The TN8-19-07 peptide is shown in boldface and the glycine and alanine linkers in italics.

FIG. 3C shows the amino acid sequence (SEQ ID NO: 615) of an Fc internal peptibody referred to hereinafter as Fc-loop-EMP. This peptibody incorporates an EPO-mimetic peptide (SEQ ID NO: 2). The inserted peptide is shown in boldface and the glycine linkers in italics. The cysteines that form a disulfide bond are underlined.

FIG. 3D shows the amino acid sequence (SEQ ID NO: 616) of an Fc internal peptibody referred to hereinafter as Fc-loop-Amp2. Bioactive peptide (SEQ ID NO: 28) is highlighted in boldface and glycine linkers in italics. There is no disulfide constraint in the AMP-2 peptide insertion.

FIG. 3E shows the amino acid sequence (SEQ ID NO: 617) of a C-terminally linked peptibody referred to hereinafter as Fc-loop-AMP2-dimer. This tandem-linked therapeutic peptide dimer shows the therapeutic peptide sequence (SEQ ID NO: 28) in boldface and the linkers in italics. This molecule incorporates a tandem peptide dimer of the same peptide sequence as found in Fc-loop-AMP-2.

FIG. 10B shows the amino acid sequence (SEQ ID NO: 619) of a C-terminally linked peptibody referred to herein as TN8-Con4. This molecule incorporates the same peptide sequence as Fc-loop-ang2 (SEQ ID NO: 147). The bioactive peptide is highlighted in boldface and the glycine and alanine linkers in italics.

FIG. 11 shows the expression and distribution in E. coli of the Fc-loop TN8-Con4 (#6888) and carboxy-terminal TN8-Con4 (#5564) peptibodies by SDS-PAGE. Samples of the crude cell lysate (lys), the insoluble fraction (insol) and the soluble (sol) fraction for both the Fc-loop-Tn8-Con4 (#6888) and TN8-Con4 (#5564) are shown in reducing gels.

FIG. 16 shows the results of an in vitro enzyme-linked immunosorbent assay (ELISA) for Fc-loop-ang2, TN8-Con4, and Fc-ang2-tandem.

FIG. 17 shows the results of a UT7 erythropoietin proliferation assay for Fc-loop-EMP. In the assay, the activity of two different of Fc-loop-EMP molecules is compared to that of epoetin alfa.

FIG. 18 shows the expression and distribution in E. coli of the Fc-loop TN8-Amp2 (#6875) peptibody by SDS-PAGE. Samples of the crude cell lysate (lys), the insoluble fraction (insol) and the soluble (sol) fraction for the Fc-loop-Amp2 (#6888) are shown in reducing gels.

The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

When used in connection with an amino acid sequence, the term “comprising” means that a compound may include additional amino acids on either or both of the N- or C-termini of the given sequence.

“Antibody” or “antibody peptide(s)” refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies. In certain embodiments, binding fragments are produced by recombinant DNA techniques. In additional embodiments, binding fragments are produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv, and single-chain antibodies.

The term “native Fc” refers to a molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form, into which a peptide sequence may be added by insertion into or replacement of a loop region. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.

The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published 25 Sep. 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference. Thus, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.

The term “multimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. Multimers may be formed by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Exemplary dimers within the scope of this invention are as shown in U.S. Pat. No. 6,660,843, FIG. 2, which is hereby incorporated by reference.

The terms “derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR1, NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R1 and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R2 or —NR3R4 wherein R2, R3 and R4 are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.

The term “polypeptide” refers to molecules of greater than 40 amino acids, whether existing in nature or not, provided that such molecules are not membrane-bound. Exemplary polypeptides include IL-1ra, leptin, soluble TNF receptors type 1 and type 2 (sTNF-R1, sTNF-R2), KGF, EPO, TPO, G-CSF, darbepoietin, Fab fragments and the like.

The term “peptide” refers to molecules of 2 to 40 amino acids, with molecules of 3 to 20 amino acids preferred and those of 6 to 15 amino acids most preferred. Exemplary peptides may be randomly generated by any of the methods cited above, carried in a peptide library (e.g., a phage display library), or derived by digestion of proteins.

The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E. coli display, ribosome display, yeast-based screening, RNA-peptide screening, chemical screening, rational design, protein structural analysis, and the like.

The term “pharmacologically active” means that a substance so described is determined to have activity that affects a medical parameter (e.g., blood pressure, blood cell count, cholesterol level) or disease state (e.g., cancer, autoimmune disorders). Thus, pharmacologically active peptides comprise agonistic or mimetic and antagonistic peptides as defined below.

The terms “-mimetic peptide” and “-agonist peptide” refer to a peptide having biological activity comparable to a protein (e.g., EPO, TPO, G-CSF) that interacts with a protein of interest. These terms further include peptides that indirectly mimic the activity of a protein of interest, such as by potentiating the effects of the natural ligand of the protein of interest; see, for example, the G-CSF-mimetic peptides listed in Table 2 hereof and in Table 7 of U.S. Pat. No. 6,660,843, which is hereby incorporated by reference. Thus, the term “EPO-mimetic peptide” comprises any peptides that can be identified or derived as described in Wrighton et al. (1996), Science 273: 458-63, Naranda et al. (1999), Proc. Natl. Acad. Sci. USA 96: 7569-74, or any other reference in Table 2 identified as having EPO-mimetic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The term “-antagonist peptide” or “inhibitor peptide” refers to a peptide that blocks or in some way interferes with the biological activity of the associated protein of interest, or has biological activity comparable to a known antagonist or inhibitor of the associated protein of interest. Thus, the term “TNF-antagonist peptide” comprises peptides that can be identified or derived as described in Takasaki et al. (1997), Nature Biotech. 15: 1266-70 or any of the references in Table 2 identified as having TNF-antagonistic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The term “TPO-mimetic peptide” comprises peptides that can be identified or derived as described in Cwirla et al. (1997), Science 276: 1696-9, U.S. Pat. Nos. 5,869,451 and 5,932,946; U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003; WO 03/031589, published Apr. 17, 2003 and any other reference in Table 2 identified as having TPO-mimetic subject matter, as well as WO 00/24770, published May 4, 2000, which is hereby incorporated by reference. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The term “ang-2-binding peptide” comprises peptides that can be identified or derived as described in U.S. Pat. App. No. 2003/0229023, published Dec. 11, 2003; WO 03/057134, published Jul. 7, 2003; U.S. 2003/0236193, published Dec. 25, 2003; and any other reference in Table 2 identified as having subject matter related to ang-2. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The term “NGF-binding peptide” comprises peptides that can be identified or derived as described in WO 04/026329, published Apr. 1, 2004 and any other reference in Table 2 identified as having subject matter related to NGF. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The term “myostatin-binding peptide” comprises peptides that can be identified or derived as described in U.S. Ser. No. 10/742,379, filed Dec. 19, 2003 and any other reference in Table 2 identified as having subject matter related to myostatin. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

Additionally, physiologically acceptable salts of the compounds of this invention are also encompassed herein. By “physiologically acceptable salts” is meant any salts that are known or later discovered to be pharmaceutically acceptable. Some specific examples are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate; and oxalate.

Structure of Compounds

In General

In the compositions of matter prepared in accordance with this invention, the peptide may be attached to the vehicle through the peptide's N-terminus or C-terminus. Thus, the vehicle-peptide molecules of this invention may be described by the following formula I:
(X1)a—F1—(X2)b I
wherein:

F1 is an Fc domain modified so that it comprises at least one X3 in a loop region;

X1 and X2 are each independently selected from -(L1)c-P1, -(L1)c-P1-(L2)d-P2, -(L1)c-P1-(L2)d-P2-(L3)e-P3, and -(L1)c-P1-(L2)d-P2-(L3)e-P3-(L4)f-P4;

X3 is independently selected from -(L5)c-P5, -(L5)c-P5-(L6)d-P6, -(L5)c-P5-(L6)d-P6-(L7)e-P7, and -(L5)c-P5-(L6)d-P6-(L7)e-P7-(L8)f-P8;

In preferred embodiments, a and b are both zero—i.e., neither X1 nor X2 groups appear at the N-terminus or C-terminus of the Fc domain.

Those of ordinary skill in the art will appreciate that more than one X3 substituent may be present in the Fc domain, and that the multiple X3 substituents may be different; for example, comprising different P5 peptides, different linkers attached to the same peptide sequence, and so on. Likewise, X1 and X2 may be the same or different, and the integers c through f may be different for X1, X2, and X3.

Thus, compound I comprises compounds of the formulae
X1—F1 II
and multimers thereof wherein F1 is attached at the C-terminus of X1;
F1—X2 III
and multimers thereof wherein F1 is attached at the N-terminus of X2;
F1-(L1)c-P1 IV
and multimers thereof wherein F1 is attached at the N-terminus of —(L1)c-P1; and
F1-(L1)c-P1-(L2)d-P2 V
and multimers thereof wherein F1 is attached at the N-terminus of -L1-P1-L2-P2.

Peptides

Any number of peptides may be used in conjunction with the present invention. Preferred peptides bind to angiopoietin-2 (ang-2), myostatin, nerve growth factor (NGF), tumor necrosis factor (TNF), B cell activating factor (BAFF, also referred to as TALL-1) or mimic the activity of EPO, TPO, or G-CSF. Targeting peptides are also of interest, including tumor-homing peptides, membrane-transporting peptides, and the like. All of these classes of peptides may be discovered by methods described in the references cited in this specification and other references.

Phage display, in particular, is useful in generating peptides for use in the present invention. It has been stated that affinity selection from libraries of random peptides can be used to identify peptide ligands for any site of any gene product. Dedman et al. (1993), J. Biol. Chem. 268: 23025-30. Phage display is particularly well suited for identifying peptides that bind to such proteins of interest as cell surface receptors or any proteins having linear epitopes. Wilson et al. (1998), Can. T. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins are extensively reviewed in Herz et al. (1997), J. Receptor & Signal Transduction Res. 17(5): 671-776, which is hereby incorporated by reference. Such proteins of interest are preferred for use in this invention.

A particularly preferred group of peptides are those that bind to cytokine receptors. Cytokines have recently been classified according to their receptor code. See Inglot (1997), Archivum Immunologiae et Therapiae Experimentalis 45: 353-7, which is hereby incorporated by reference. Among these receptors, most preferred are the CKRs (family I in Table 3). The receptor classification appears in Table 3.

TABLE 3

Cytokine Receptors Classified by Receptor Code

Cytokines (ligands)

Receptor Type

family

subfamily

family

subfamily

I.

Hematopoietic

1.

IL-2, IL-4, IL-7,

I.

Cytokine R

1.

shared γCr, IL-

cytokines

IL-9, IL-13, IL-

(CKR)

9R, IL-4R

15

2.

shared GP 140

2.

IL-3, IL-5, GM-

βR

CSF

3.

3.shared RP

3.

IL-6, IL-11, IL-

130, IL-6 R,

12, LIF, OSM,

Leptin R

CNTF, Leptin

4.

“single chain”

(OB)

R, GCSF-R,

4.

G-CSF, EPO,

TPO-R, GH-R

TPO, PRL, GH

5.

other Rb

5.

IL-17, HVS-IL-

17

II.

IL-10 ligands

IL-10, BCRF-1,

II.

IL-10 R

HSV-IL-10

III.

Interferons

1.

IFN-α1, α2, α4,

III.

Interferon R

1.

IFNAR

m, t, IFN-βc

2.

IFNGR

2.

IFN-γ

IV.

IL-1 and IL-1

1.

IL-1α, IL-1β,

IV.

IL-1R

1.

IL-1R, IL-

like ligands

IL-1Ra

1RAcP

2.

IL-18, IL-18BP

2.

IL-18R, IL-

18RAcP

V.

TNF family

1.

TNF-α, TNF-β

3.

NGF/TNF Rd

TNF-RI, AGP-3R,

(LT), FASL,

DR4, DR5, OX40,

CD40 L,

OPG, TACI, CD40,

CD30L, CD27

FAS, ODR

L, OX40L,

OPGL, TRAIL,

APRIL, AGP-3,

BLys, TL5,

Ntn-2, KAY,

Neutrokine-α

VI.

Chemokines

1.

α chemokines:

4.

Chemokine R

1.

CXCR

IL-8, GRO α, β,

2.

CCR

γ, IF-10, PF-4,

3.

CR

SDF-1

4.

DARCe

2.

β chemokines:

MIP1α, MIP1β,

MCP-1,2,3,4,

RANTES,

eotaxin

3.

γ chemokines:

lymphotactin

VII.

Growth factors

1.1

SCF, M-CSF,

VII.

RKF

1.

TK sub-family

PDGF-AA, AB,

1.1

IgTK III R,

BB, KDR, FLT-

VEGF-RI,

1, FLT-3L,

VEGF-RII,

VEGF, SSV-

1.2

IgTK IV R

PDGF, HGF, SF

1.3

Cysteine-rich

1.2

FGFα, FGFβ

TK-I

1.3

EGF, TGF-α,

1.4

Cysteine rich

VV-F19 (EGF-

TK-II, IGF-RI

like)

1.5

Cysteine knot

1.4

IGF-I, IGF-II,

TK V

Insulin

2.

Serine-

1.5

NGF, BDNF,

threonine

NT-3, NT-4f

kinase

2.

TGF-β1, β2, β3

subfamily

(STKS)g

1IL-17R - belongs to CKR family but is unassigned to 4 indicated subfamilies.

2Other IFN type I subtypes remain unassigned. Hematopoietic cytokines, IL-10 ligands and interferons do not possess functional intrinsic protein kinases. The signaling molecules for the cytokines are JAK's, STATs and related non-receptor molecules. IL-14, IL-16 and IL-18 have been cloned but according to the receptor code they remain unassigned.

3TNF receptors use multiple, distinct intracellular molecules for signal transduction including “death domain” of FAS R and 55 kDa TNF-αR that participates in their cytotoxic effects. NGF/TNF R can bind both NGF and related factors as well as TNF ligands. Chemokine receptors are seven transmembrane (7TM, serpentine) domain receptors. They are G protein-coupled.

4The Duffy blood group antigen (DARC) is an erythrocyte receptor that can bind several different chemokines. IL-1R belongs to the immunoglobulin superfamily but their signal transduction events characteristics remain unclear.

6STKS may encompass many other TGF-β-related factors that remain unassigned. The protein kinases are intrinsic part of the intracellular domain of receptor kinase family (RKF). The enzymes participate in the signals transmission via the receptors.

Particular proteins of interest as targets for peptide generation in the present invention include the following:

Exemplary peptides for this invention appear in Tables 4 through 20 of U.S. Pat. No. 6,660,843, which are hereby incorporated by reference. Additional preferred peptides appear in U.S. 2003/0229023, published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S. 2003/0236193, published Dec. 25,2003; WO 00/24770, published May 4, 2000; U.S. 2003/0176352, published Sep. 18, 2003; WO 03/031589, published Apr. 17, 2003; U.S. Ser. No. 10/666,480, filed Sep. 18, 2003; WO 04/026329, published Apr. 1, 2004; U.S. Ser. No. 10/742,379, filed Dec. 19, 2003; PCT/US03/40781, filed Dec. 19, 2003, each of which are hereby incorporated by reference. Such peptides may be prepared by methods disclosed in the art.

Particularly preferred peptides appear in the tables below. Single letter amino acid abbreviations are used. Any of these peptides may be linked in tandem (i.e., sequentially), with or without linkers. Any peptide containing a cysteinyl residue may be cross-linked with another Cys-containing peptide or protein. Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well. Any of these peptides may be derivatized as described herein. All peptides are linked through peptide bonds unless otherwise noted.

TABLE 4

EPO-mimetic peptide sequences

SEQUENCE

SEQ ID NO:

YXCXXGPXTWXCXP

1

GGTYSCHFGPLTWVCKPQGG

2

GGDYHCRMGPLTWVCKPLGG

3

GGVYACRMGPITWVCSPLGG

4

VGNYMCHFGPITWVCRPGGG

5

GGLYLCRFGPVTWDCGYKGG

6

GGTYSCHFGPLTWVCKPQGGSSK

7

GGTYSCHGPLTWVCKPQGG

8

VGNYMAHMGPITWVCRPGG

9

GGPHHVYACRMGPLTWIC

10

GGTYSCHFGPLTWVCKPQ

11

GGLYACHMGPMTWVCQPLRG

12

TIAQYICYMGPETWECRPSPKA

13

YSCHFGPLTWVCK

14

YCHFGPLTWVC

15

GGLYLCRFGPVTWDCGYKGG

16

GGTYSCHFGPLTWVCKPQGG

17

GGDYHCRMGPLTWVCKPLGG

18

VGNYMCHFGPITWVCRPGGG

19

GGVYACRMGPITWVCSPLGG

20

VGNYMAHMGPITWVCRPGG

21

GGTYSCHFGPLTWVCKPQ

22

GGLYACHMGPMTWVCQPLRG

23

TIAQYICYMGPETWECRPSPKA

24

YSCHFGPLTWVCK

25

YCHFGPLTWVC

26

SCHFGPLTWVCK

27

TABLE 5

TPO-mimetic peptide sequences

SEQUENCE

SEQ ID NO:

IEGPTLRQWLAARA

28

IEGPTLRQWLAAKA

29

IEGPTLREWLAARA

30

TLREWL

31

GRVRDQVAGW

32

GRVKDQIAQL

33

GVRDQVSWAL

34

ESVREQVMKY

35

SVRSQISASL

36

GVRETVYRHM

37

GVREVIVMHML

38

GRVRDQIWAAL

39

AGVRDQILIWL

40

GRVRDQIMLSL

41

CTLRQWLQGC

42

CTLQEFLEGC

43

CTRTEWLHGC

44

CTLREWLHGGFC

45

CTLREWVFAGLC

46

CTLRQWLILLGMC

47

CTLAEFLASGVEQC

48

CSLQEFLSHGGYVC

49

CTLREFLDPTTAVC

50

CTLKEWLVSHEVWC

51

REGPTLRQWM

52

EGPTLRQWLA

53

ERGPFWAKAC

54

REGPRCVMWM

55

CGTEGPTLSTWLDC

56

CEQDGPTLLEWLKC

57

CELVGPSLMSWLTC

58

CLTGPFVTQWLYEC

59

CRAGPTLLEWLTLC

60

CADGPTLREWISFC

61

GGCTLREWLHGGFCGG

62

GGCADGPTLREWISFCGG

63

GNADGPTLRQWLEGRRPKN

64

LAIEGPTLRQWLHGNGRDT

65

HGRVGPTLREWKTQVATKK

66

TIKGPTLRQWLKSREHTS

67

ISDGPTLKEWLSVTRGAS

68

SIEGPTLREWLTSRTPHS

69

GAREGPTLRQWLEWVRVG

70

RDLDGPTLRQWLPLPSVQ

71

ALRDGPTLKQWLEYRRQA

72

ARQEGPTLKEWLFWVRMG

73

EALLGPTLREWLAWRRAQ

74

MARDGPTLREWLRTYRMM

75

WMPEGPTLKQWLFHGRGQ

76

HIREGPTLRQWLVALRMV

77

QLGHGPTLRQWLSWYRGM

78

ELRQGPTLHEWLQHLASK

79

VGIEGPTLRQWLAQRLNP

80

WSRDGPTLREWLAWRAVG

81

AVPQGPTLKQWLLWRRCA

82

RIREGPTLKEWLAQRRGF

83

RFAEGPTLREWLEQRKLV

84

DRFQGPTLREWLAAIRSV

85

AGREGPTLREWLNMRVWQ

86

ALQEGPTLRQWLGWGQWG

87

YCDEGPTLKQWLVCLGLQ

88

WCKEGPTLREWLRWGFLC

89

CSSGGPTLREWLQCRRMQ

90

CSWGGPTLKQWLQCVRAK

91

CQLGGPTLREWLACRLGA

92

CWEGGPTLKEWLQCLVER

93

CRGGGPTLHQWLSCFRWQ

94

CRDGGPTLRQWLACLQQK

95

ELRSGPTLKEWLVWRLAQ

96

GCRSGPTLREWLACREVQ

97

TCEQGPTLRQWLLCRQGR

98

QGYCDEGPTLKQWLVCLGLQHS

99

TABLE 6

Ang-2 binding peptide sequences

SEQUENCE

SEQ ID NO.

WDPWT

100

WDPWTC

101

Cz2WDPWT (wherein z2 is an acidic or

102

neutral polar amino acid residue)

Cz2WDPWTC (wherein z2 is an acidic or

103

neutral polar amino acid residue)

PIRQEECDWDPWTCEHMWEV

104

TNIQEECEWDPWTCDHMPGK

105

WYEQDACEWDPWTCEHMAEV

106

NRLQEVCEWDPWTCEHMENV

107

AATQEECEWDPWTCEHMPRS

108

LRHQEGCEWDPWTCEHMFDW

109

VPRQKDCEWDPWTCEHMYVG

110

SISHEECEWDPWTCEHMQVG

111

WAAQEECEWDPWTCEHMGRM

112

TWPQDKCEWDPWTCEHMGST

113

GHSQEECGWDPWTCEHMGTS

114

QHWQEECEWDPWTCDHMPSK

115

NVRQEKCEWDPWTCEHMPVR

116

KSGQVECNWDPWTCEHMPRN

117

VKTQEHCDWDPWTCEHMREW

118

AWGQEGCDWDPWTCEHMLPM

119

PVNQEDCEWDPWTCEHMPPM

120

RAPQEDCEWDPWTCAHMDIK

121

HGQNMECEWDPWTCEHMFRY

122

PRLQEECVWDPWTCEHMPLR

123

RTTQEKCEWDPWTCEHMESQ

124

QTSQEDCVWDPWTCDHMVSS

125

QVIGRPCEWDPWTCEHLEGL

126

WAQQEECAWDPWTCDHMVGL

127

LPGQEDCEWDPWTCEHMVRS

128

PMNQVECDWDPWTCEHMPRS

129

FGWSHGCEWDPWTCEHMGST

130

KSTQDDCDWDPWTCEHMVGP

131

GPRISTCQWDPWTCEHMDQL

132

STIGDMCEWDPWTCAHMQVD

133

VLGGQGCEWDPWTCRLLQGW

134

VLGGQGCQWDPWTCSHLEDG

135

TTIGSMCEWDPWTCAHMQGG

136

TKGKSVCQWDPWTCSHMQSG

137

TTIGSMCQWDPWTCAHMQGG

138

WVNEVVCEWDPWTCNHWDTP

139

VVQVGMCQWDPWTCKHMRLQ

140

AVGSQTCEWDPWTCAHLVEV

141

QGMKMFCEWDPWTCAHIVYR

142

TTIGSMCQWDPWTCEHMQGG

143

TSQRVGCEWDPWTCQHLTYT

144

QWSWPPCEWDPWTCQTVWPS

145

GTSPSFCQWDPWTCSHMVQG

146

QEECEWDPWTCEHM

147

QNYKPLDELDATLYEHFIFHYT

148

LNFTPLDELEQTLYEQWTLQQS

149

TKFNPLDELEQTLYEQWTLQHQ

150

VKFKPLDALEQTLYEHWMFQQA

151

VKYKPLDELDEILYEQQTFQER

152

TNFMPMDDLEQRLYEQFILQQG

153

SKFKPLDELEQTLYEQWTLQHA

154

QKFQPLDELEQTLYEQFMLQQA

155

QNFKPMDELEDTLYKQFLFQHS

156

YKFTPLDDLEQTLYEQWTLQHV

157

QEYEPLDELDETLYNQWMFHQR

158

SNFMPLDELEQTLYEQFMLQHQ

159

QKYQPLDELDKTLYDQFMLQQG

160

QKFQPLDELEETLYKQWTLQQR

161

VKYKPLDELDEWLYHQFTLHHQ

162

QKFMPLDELDEILYEQFMFQQS

163

QTFQPLDDLEEYLYEQWIRRYH

164

EDYMPLDALDAQLYEQFILLHG

165

HTFQPLDELEETLYYQWLYDQL

166

YKFNPMDELEQTLYEEFLFQHA

167

TNYKPLDELDATLYEHWILQHS

168

QKFKPLDELEQTLYEQWTLQQR

169

TKFQPLDELDQTLYEQWTLQQR

170

TNFQPLDELDQTLYEQWTLQQR

171

KFNPLDELEETLYEQFTFQQ

172

AGGMRPYDGMLGWPNYDVQA

173

QTWDDPCMHILGPVTWRRCI

174

APGQRPYDGMLGWPTYQRIV

175

SGQLRPCEEIFGCGTQNLAL

176

FGDKRPLECMFGGPIQLCPR

177

GQDLRPCEDMFGCGTKDWYG

178

KRPCEEIFGGCTYQ

179

GFEYCDGMEDPFTFGCDKQT

180

KLEYCDGMEDPFTQGCDNQS

181

LQEWCEGVEDPFTFGCEKQR

182

AQDYCEGMEDPFTFGCEMQK

183

LLDYCEGVQDPFTFGCENLD

184

HQEYCEGMEDPFTFGCEYQG

185

MLDYCEGMDDPFTFGCDKQM

186

LQDYCEGVEDPFTFGCENQR

187

LQDYCEGVEDPFTFGCEKQR

188

FDYCEGVEDPFTFGCDNH

189

TABLE 7

NGF-Binding Peptide Sequences

SEQUENCE

SEQ ID NO.

TGYTEYTEEWPMGFGYQWSF

190

TDWLSDFPFYEQYFGLMPPG

191

FMRFPNPWKLVEPPQGWYYG

192

VVKAPHFEFLAPPHFHEFPF

193

FSYIWIDETPSNIDRYMLWL

194

VNFPKVPEDVEPWPWSLKLY

195

TWHPKTYEEFALPFFVPEAP

196

WHFGTPYIQQQPGVYWLQAP

197

VWNYGPFFMNFPDSTYFLHE

198

WRIHSKPLDYSHVWFFPADF

199

FWDGNQPPDILVDWPWNPPV

200

FYSLEWLKDHSEFFQTVTEW

201

QFMELLKFFNSPGDSSHHFL

202

TNVDWISNNWEHMKSFFTED

203

PNEKPYQMQSWFPPDWPVPY

204

WSHTEWVPQVWWKPPNHFYV

205

WGEWINDAQVHMHEGFISES

206

VPWEHDHDLWEIISQDWHIA

207

VLHLQDPRGWSNFPPGVLEL

208

IHGCWFTEEGCVWQ

209

YMQCQFARDGCPQW

210

KLQCQYSESGCPTI

211

FLQCEISGGACPAP

212

KLQCEFSTSGCPDL

213

KLQCEFSTQGCPDL

214

KLQCEFSTSGCPWL

215

IQGCWFTEEGCPWQ

216

SFDCDNPWGHVLQSCFGF

217

SFDCDNPWGHKLQSCFGF

218

TABLE 8

Myostatin binding peptide sequences

SEQUENCE

SEQ ID NO:

KDKCKMWHWMCKPP

647

KDLCAMWHWMCKPP

219

KDLCKMWKWMCKPP

220

KDLCKMWHWMCKPK

221

WYPCYEFHFWCYDL

222

WYPCYEGHFWCYDL

223

IFGCKWWDVQCYQF

224

IFGCKWWDVDCYQF

225

ADWCVSPNWFCMVM

226

HKFCPWWALFCWDF

227

KDLCKMWHWMCKPP

228

IDKCAIWGWMCPPL

229

WYPCGEFGMWCLNV

230

WFTCLWNCDNE

231

HTPCPWFAPLCVEW

232

KEWCWRWKWMCKPE

233

FETCPSWAYFCLDI

234

AYKCEANDWGCWWL

235

NSWCEDOWHRCWWL

236

WSACYAGHFWCYDL

237

ANWCVSPNWFCMVM

238

WTECYQQEFWCWNL

239

ENTCERWKWMCPPK

240

WLPCHQEGFWCMNF

241

STMCSQWHWMCNPF

242

IFGCHWWDVDCYQF

243

IYGCKWWDIQCYDI

244

PDWCIDPDWWCKFW

245

QGHCTRWPWMCPPY

246

WQECYREGFWCLQT

247

WFDCYGPGFKCWSP

248

GVRCPKGHLWCLYP

249

HWACGYWPWSCKWV

250

GPACHSPWWWCVFG

251

TTWCISPMWFCSQQ

252

HKFCPPWAIFCWDF

253

PDWCVSPRWYCNMW

254

VWKCHWFGMDCEPT

255

KKHCQIWTWMCAPK

256

WFQCGSTLFWCYNL

257

WSPCYDHYFYCYTI

258

SWMCGFFKEVCMWV

259

EMLCMIHPVFCNPH

260

LKTCNLWPWMCPPL

261

VVGCKWYEAWCYNK

262

PIHCTQWAWMCPPT

263

DSNCPWYFLSCVIF

264

HIWCNLAMMKCVEM

265

NLQCIYFLGKCIYF

266

AWRCMWFSDVCTPG

267

WFRCFLDADWCTSV

268

EKICQMWSWMCAPP

269

WFYCHLNKSECTEP

270

FWRCAIGIDKCKRV

271

NLGCKWYEVWCFTY

272

IDLCNMWDGMCYPP

273

EMPCNIWGWMCPPV

274

WFRCVLTGIVDWSECFGL

275

GFSCTFGLDEFYVDCSPF

276

LPWCHDQVNADWGFCMLW

277

YPTCSEKFWIYGQTCVLW

278

LGPCPIHHGPWPQYCVYW

279

PFPCETHQISWLGHCLSF

280

HWGCEDLMWSWHPLCRRP

281

LPLCDADMMPTIGFCVAY

282

SHWCETTFWMNYAKCVHA

283

LPKCTHVPFDQGGFCLWY

284

FSSCWSPVSRQDMFCVFY

285

SHKCEYSGWLQPLCYRP

286

PWWCQDNYVQHMLHCDSP

287

WFRCMLMNSFDAFQCVSY

288

PDACRDQPWYMFMGCMLG

289

FLACFVEFELCFDS

290

SAYCIITESDPYVLCVPL

291

PSICESYSTMWLPMCQHN

292

WLDCHDDSWAWTKMCRSH

293

YLNCVMMNTSPFVECVFN

294

YPWCDGFMIQQGITCMFY

295

FDYCTWLNGFKDWKCWSR

296

LPLCNLKEISHVQACVLF

297

SPECAFARWLGIEQCQRD

298

YPQCFNLHLLEWTECDWF

299

RWRCEIYDSEFLPKCWFF

300

LVGCDNVWHRCKLF

301

AGWCHVWGEMFGMGCSAL

302

HHECEWMARWMSLDCVGL

303

FPMCGIAGMKDFDFCVWY

304

RDDCTFWPEWLWKLCERP

305

YNFCSYLFGVSKEACQLP

306

AHWCEQGPWRYGNICMAY

307

NLVCGKISAWGDEACARA

308

HNVCTIMGPSMKWFCWND

309

NDLCAMWGWRNTIWCQNS

310

PPFCQNDNDMLQSLCKLL

311

WYDCNVPNELLSGLCRLF

312

YGDCDQNHWMWPFTCLSL

313

GWMCHFDLHDWGATCQPD

314

YFHCMFGGHEFEVHCESF

315

AYWCWHGQCVRF

316

SEHWTFTDWDGNEWWVRPF

317

MEMLDSLFELLKDMVPISKA

318

SPPEEALMEWLGWQYGKFT

319

SPENLLNDLYILMTKQEWYG

320

FHWEEGIPFHVVTPYSYDRM

321

KRLLEQFMNDLAELVSGHS

322

DTRDALFQEFYEFVRSRLVI

323

RMSAAPRPLTYRDIMDQYWH

324

NDKAHFFEMFMFDVHNFVES

325

QTQAQKIDGLWELLQSIRNQ

326

MLSEFEEFLGNLVHRQEA

327

YTPKMGSEWTSFWHNRIHYL

328

LNDTLLRELKMVLNSLSDMK

329

FDVERDLMRWLEGFMQSAAT

330

HHGWNYLRKGSAPQWFEAWV

331

VESLHQLQMWLDQKLASGPH

332

RATLLKDFWQLVEGYGDN

333

EELLREFYRFVSAFDY

334

GLLDEFSHFIAEQFYQMPGG

335

YREMSMLEGLLDVLERLQHY

336

HNSSQMLLSELIMLVGSMMQ

337

WREHFLNSDYIRDKLIAIDG

338

QFPFYVFDDLPAQLEYWIA

339

EFFHWLHNHRSEVNHWLDMN

340

EALFQNFFRDVLTLSEREY

341

QYWEQQWMTYFRENGLHVQY

342

NQRMMLEDLWRIMTPMFGRS

343

FLDELKAELSRHYALDDLDE

344

GKLIEGLLNELMQLETFMPD

345

ILLLDEYKKDWKSWF

346

QGHCTRWPWMCPPYGSGSATGGSGST

347

ASSGSGSATGQGHCTRWPWMCPPY

WYPCYEGHFWCYDLGSGSTASSGSGSA

348

TGWYPCYEGHFWCYDL

HTPCPWFAPLCVEWGSGSATGGSGSTA

349

SSGSGSATGHTPCPWFAPLCVEW

PDWCIDPDWWCKFWGSGSATGGSGST

350

ASSGSGSATGPDWCIDPDWWCKFW

ANWCVSPNWFCMVMGSGSATGGSGST

351

ASSGSGSATGANWCVSPNWFCMVM

PDWCIDPDWWCKFWGSGSATGGSGST

352

ASSGSGSATGPDWCIDPDWWCKFW

HWACGYWPWSCKWVGSGSATGGSGST

353

ASSGSGSATGHWACGYWPWSCKWV

KKHCQIWTWMCAPKGSGSATGGSGST

354

ASSGSGSATGQGHCTRWPWMCPPY

QGHCTRWPWMCPPYGSGSATGGSGST

355

ASSGSGSATGKKHCQIWTWMCAPK

KKHCQIWTWMCAPKGSGSATGGSGST

356

ASSGSGSATGQGHCTRWPWMCPPY

KKHCQIWTWMCAPKGGGGGGGGQGH

357

CTRWPWMCPPY

QGHCTRWPWMCPPYGGGGGGKKHCQ

358

IWTWMCAPK

VALHGQCTRWPWMCPPQREG

359

YPEQGLCTRWPWMCPPQTLA

360

GLNQGHCTRWPWMCPPQDSN

361

MITQGQCTRWPWMCPPQPSG

362

AGAQEHCTRWPWMCAPNDWI

363

GVNQGQCTRWRWMCPPNGWE

364

LADHGQCIRWPWMCPPEGWE

365

ILEQAQCTRWPWMCPPQRGG

366

TQTHAQCTRWPWMCPPQWEG

367

VVTQGHCTLWPWMCPPQRWR

368

IYPHDQCTRWPWMCPPQPYP

369

SYWQGQCTRWPWMCPPQWRG

370

MWQQGHCTRWPWMCPPQGWG

371

EFTQWHCTRWPWMCPPQRSQ

372

LDDQWQCTRWPWMCPPQGFS

373

YQTQGLCTRWPWMCPPQSQR

374

ESNQGQCTRWPWMCPPQGGW

375

WTDRGPCTRWPWMCPPQANG

376

VGTQGQCTRWPWMCPPYETG

377

PYEQGKCTRWPWMCPPYEVE

378

SEYQGLCTRWPWMCPPQGWK

379

TFSQGHCTRWPWMCPPQGWG

380

PGAHDHCTRWPWMCPPQSRY

381

VAEEWHCRRWPWMCPPQDWR

382

VGTQGHCTRWPWMCPPQPAG

383

EEDQAHCRSWPWMCPPQGWV

384

ADTQGHCTRWPWMCPPQHWF

385

SGPQGHCTRWPWMCAPQGWF

386

TLVQGHCTRWPWMCPPQRWV

387

GMAHGKCTRWAWMCPPQSWK

388

ELYHGQCTRWPWMCPPQSWA

389

VADHGHCTRWPWMCPPQGWG

390

PESQGHCTRWPWMCPPQGWG

391

IPAHGHCTRWPWMCPPQRWR

392

FTVHGHCTRWPWMCPPYGWV

393

PDFPGHCTRWRWMCPPQGWE

394

QLWQGPCTQWPWMCPPKGRY

395

HANDGHCTRWQWMCPPQWGG

396

ETDHGLCTRWPWMCPPYGAR

397

GTWQGLCTRWPWMCPPQGWQ

398

VATQGQCTRWPWMCPPQGWG

399

VATQGQCTRWPWMCPPQRWG

400

QREWYPCYGGHLWCYDLHKA

401

ISAWYSCYAGHFWCWDLKQK

402

WTGWYQCYGGHLWCYDLRRK

403

KTFWYPCYDGHFWCYNLKSS

404

ESRWYPCYEGHLWCFDLTET

405

MEMLDSLFELLKDMVPISKA

406

RMEMLESLLELLKEIVPMSKAG

407

RMEMLESLLELLKEIVPMSKAR

408

RMEMLESLLELLKDIVPMSKPS

409

GMEMLESLFELLQEIVPMSKAP

410

RMEMLESLLELLKDIVPISNPP

411

RIEMLESLLELLQEIVPISKAE

412

RMEMLQSLLELLKDIVPMSNAR

413

RMEMLESLLELLKEIVPTSNGT

414

RMEMLESLFELLKEIVPMSKAG

415

RMEMLGSLLELLKEIVPMSKAR

416

QMELLDSLFELLKEIVPKSQPA

417

RMEMLDSLLELLKEIVPMSNAR

418

RMEMLESLLELLHEIVPMSQAG

419

QMEMLESLLQLLKEIVPMSKAS

420

RMEMLDSLLELLKDMVPMTTGA

421

RIEMLESLLELLKDMVPMANAS

422

RMEMLESLLQLLNEIVPMSRAR

423

RMEMLESLFDLLKELVPMSKGV

424

RIEMLESLLELLKDIVPIQKAR

425

RMELLESLFELLKDMVPMSDSS

426

RMEMLESLLEVLQEIVPRAKGA

427

RMEMLDSLLQLLNEIVPMSHAR

428

RMEMLESLLELLKDIVPMSNAG

429

RMEMLQSLFELLKGMVPISKAG

430

RMEMLESLLELLKEIVPNSTAA

431

RMEMLQSLLELLKEIVPISKAG

432

RIEMLDSLLELLNELVPMSKAR

433

HHGWNYLRKGSAPQWFEAWV

434

QVESLQQLLMWLDQKLASGPQG

435

RMELLESLFELLKEMVPRSKAV

436

QAVSLQHLLMWLDQKLASGPQH

437

DEDSLQQLLMWLDQKLASGPQL

438

PVASLQQLLIWLDQKLAQGPHA

439

EVDELQQLLNWLDHKLASGPLQ

440

DVESLEQLLMWLDHQLASGPHG

441

QVDSLQQVLLWLEHKLALGPQV

442

GDESLQHLLMWLEQKLALGPHG

443

QIEMLESLLDLLRDMVPMSNAF

444

EVDSLQQLLMWLDQKLASGPQA

445

EDESLQQLLIYLDKMLSSGPQV

446

AMDQLHQLLIWLDHKLASGPQA

447

RIEMLESLLELLDEIALIPKAW

448

EVVSLQHLLMWLEHKLASGPDG

449

GGESLQQLLMWLDQQLASGPQR

450

GVESLQQLLIFLDHMLVSGPHD

451

NVESLEHLMMWLERLLASGPYA

452

QVDSLQQLLIWLDHQLASGPKR

453

EVESLQQLLMWLEHKLAQGPQG

454

EVDSLQQLLMWLDQKLASGPHA

455

EVDSLQQLLMWLDQQLASGPQK

456

GVEQLPQLLMWLEQKLASGPQR

457

GEDSLQQLLMWLDQQLAAGPQV

458

ADDSLQQLLMWLDRKLASGPHV

459

PVDSLQQLLIWLDQKLASGPQG

460

RATLLKDFWQLVEGYGDN

461

DWRATLLKEFWQLVEGLGDNLV

462

QSRATLLKEFWQLVEGLGDKQA

463

DGRATLLTEFWQLVQGLGQKEA

464

LARATLLKEFWQLVEGLGEKVV

465

GSRDTLLKEFWQLVVGLGDMQT

466

DARATLLKEFWQLVDAYGDRMV

467

NDRAQLLRDFWQLVDGLGVKSW

468

GVRETLLYELWYLLKGLGANQG

469

QARATLLKEFCQLVGCQGDKLS

470

QERATLLKEFWQLVAGLGQNMR

471

SGRATLLKEFWQLVQGLGEYRW

472

TMRATLLKEFWLFVDGQREMQW

473

GERATLLNDFWQLVDGQGDNTG

474

DERETLLKEFWQLVHGWGDNVA

475

GGRATLLKELWQLLEGQGANLV

476

TARATLLNELVQLVKGYGDKLV

477

GMRATLLQEFWQLVGGQGDNWM

478

STRATLLNDLWQLMKGWAEDRG

479

SERATLLKELWQLVGGWGDNFG

480

VGRATLLKEFWQLVEGLVGQSR

481

EIRATLLKEFWQLVDEWREQPN

482

QLRATLLKEFLQLVHGLGETDS

483

TQRATLLKEFWQLIEGLGGKHV

484

HYRATLLKEFWQLVDGLREQGV

485

QSRVTLLREFWQLVESYRPIVN

486

LSRATLLNEFWQFVDGQRDKRM

487

WDRATLLNDFWHLMEELSQKPG

488

QERATLLKEFWRMVEGLGKNRG

489

NERATLLREFWQLVGGYGVNQR

490

YREMSMLEGLLDVLERLQHY

491

HQRDMSMLWELLDVLDGLRQYS

492

TQRDMSMLDGLLEVLDQLRQQR

493

TSRDMSLLWELLEELDRLGHQR

494

MQHDMSMLYGLVELLESLGHQI

495

WNRDMRMLESLFEVLDGLRQQV

496

GYRDMSMLEGLLAVLDRLGPQL

497

TQRDMSMLEGLLEVLDRLGQQR

498

WYRDMSMLEGLLEVLDRLGQQR

499

HNSSQMLLSELIMLVGSMMQ

500

TQNSRQMLLSDFMMLVGSMIQG

501

MQTSRHILLSEFMMLVGSIMHG

502

HDNSRQMLLSDLLHLVGTMIQG

503

MENSRQNLLRELIMLVGNMSHQ

504

QDTSRHMLLREFMMLVGEMIQG

505

DQNSRQMLLSDLMILVGSMIQG

506

EFFHWLHNHRSEVNHWLDMN

507

NVFFQWVQKHGRVVYQWLDINV

508

FDFLQWLQNHRSEVEHWLVMDV

509

TABLE 9

BAFF binding peptide sequences

SEQUENCE

SEQ ID NO:

PGTCFPFPWECTHA

510

WGACWPFPWECFKE

511

VPFCDLLTKHCFEA

512

GSRCKYKWDVLTKQCFHH

513

LPGCKWDLLIKQWVCDPL

514

SADCYFDILTKSDVCTSS

515

SDDCMYDQLTRMFICSNL

516

DLNCKYDELTYKEWCQFN

517

FHDCKYDLLTRQMVCHGL

518

RNHCFWDHLLKQDICPSP

519

ANQCWWDSLTKKNVCEFF

520

YKGRQQMWDILTRSWVVSL

521

QQDVGLWWDILTRAWMPNI

522

QQNAQRVWDLLIRTWVYPQ

523

GWNEAWWDELTKIWVLEQQ

524

RITCDTWDSLIKKCVPQQS

525

GAIMQQFWDSLTKTWLRQS

526

WLHSGWWDPLTKHWLQQKV

527

SEWFFWFDPLTRAQQLKFR

528

GVWFWWFDPLTKQWTQQAG

529

MQQCKGYYDILTKWCVTNG

530

LWSKEVWDILTKSWVSQQA

531

KAAGWWFDWLTKVWVPAP

532

AYQQTWFWDSLTRLWLSTT

533

SGQQHFWWDLLTRSWTPST

534

LGVGQQKWDPLTKQWVSRG

535

VGKMCQQWDPLIKRTVCVG

536

CRQGAKFDLLTKQCLLGR

537

GQAIRHWDVLTKQWVDSQQ

538

RGPCGSWDLLTKHCLDSQQ

539

WQWKQQQWDLLTKQMVWVG

540

PITICRKDLLTKQVVCLD

541

KTCNGKWDLLTKQCLQQQA

542

KCLKGKWDLLTKQCVTEV

543

RCWNGKWDLLTKQCIHPW

544

NRDMRKWDPLIKQWIVRP

545

QQAAAATWDLLTKQWLVPP

546

PEGGPKWDPLTKQQFLPPV

547

QQTPQQKKWDLLTKQWFTRN

548

IGSPCKWDLLTKQMICQQT

549

CTAAGKWDLLTKQCIQQEK

550

VSQCMKWDLLTKQCLQQGW

551

VWGTWKWDLLTKQYLPPQQ

552

GWWEMKWDLLTKQWYRPQQ

553

TAQQVSKWDLLTKQWLPLA

554

QLWGTKWDLLTKQYIQQIM

555

WATSQKWDLLTKQWVQQNM

556

QQRQCAKWDLLTKQCVLFY

557

KTTDCKWDLLTKQRICQQV

558

LLCQQGKWDLLTKQCLKLR

559

LMWFWKWDLLTKQLVPTF

560

QQTWAWKWDLLTKQWIGPM

561

NKELLKWDLLTKQCRGRS

562

GQQKDLKWDLLTKQYVRQS

563

PKPCQQKWDLLTKQCLGSV

564

GQIGWKWDLLTKQWIQQTR

565

VWLDWKWDLLTKQWIHPQQ

566

QQEWEYKWDLLTKQWGWLR

567

HWDSWKWDLLTKQWVVQQA

568

TRPLQQKWDLLTKQWLRVG

569

SDQWQQKWDLLTKQWFWDV

570

QQQTFMKWDLLTKQWIRRH

571

QQGECRKWDLLTKQCFPGQ

572

GQQMGWRWDPLIKMCLGPS

573

QQLDGCKWDLLTKQKVCIP

574

HGYWQQKWDLLTKQWVSSE

575

HQQGQCGWDLLTRIYLPCH

576

LHKACKWDLLTKQCWPMQQ

577

GPPGSVWDLLTKIWIQQTG

578

ITQQDWRFDTLTRLWLPLR

579

QQGGFAAWDVLTKMWITVP

580

GHGTPWWDALTRIWILGV

581

VWPWQQKWDLLTKQFVFQD

582

WQQWSWKWDLLTRQYISSS

583

NQQTLWKWDLLTKQFITYM

584

PVYQQGWWDTLTKLYIWDG

585

WLDGGWRDPLIKRSVQQLG

586

GHQQQFKWDLLTKQWVQSN

587

QQRVGQFWDVLTKMFITGS

588

QQAQGWSYDALIKTWIRWP

589

GWMHWKWDPLTKQQALPWM

590

GHPTYKWDLLTKQWILQQM

591

WNNWSLWDPLTKLWLQQQN

592

WQWGWKWDLLTKQWVQQQ

593

GQMGWRWDPLTKMWLGTS

594

Fc Domains

This invention requires the presence of at least one Fc domain modified to comprise a peptide sequence.

As noted above, both native Fc's and Fc variants are suitable Fc domains for use within the scope of this invention. A native Fc may be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478. In such Fc variants, one may remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants may be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which:

1. Sites involved in disulfide bond formation are removed. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, the cysteine-containing segment at the N-terminus may be truncated or cysteine residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl). In particular, one may truncate the N-terminal 20-amino acid segment of SEQ ID NO: 599 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 599 Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.

2. A native Fc is modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. One may also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO: 599 (FIG. 2A) is one such Fc variant.

3. A portion of the N-terminus of a native Fc is removed to prevent N-terminal heterogeneity when expressed in a selected host cell. For this purpose, one may delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.

4. One or more glycosylation sites are removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine).

5. Sites involved in interaction with complement, such as the C1q binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fc variant.

6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc may have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so may be removed.

7. The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed.

8. When the native Fc is derived from a non-human antibody, the native Fc may be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art.

Preferred Fc variants include the following. In SEQ ID NO: 599 (FIG. 2A) the leucine at position 15 may be substituted with glutamate;

the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, one or more tyrosine residues can be replaced by phenyalanine residues.

Additional Vehicles

The invention further embraces molecules covalently modified to include one or more water soluble polymer attachments, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol, as described U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337. Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers. Particularly preferred are peptibodies covalently modified with polyethylene glycol (PEG) subunits. Water-soluble polymers may be bonded at specific positions, for example at the amino terminus of the peptibodies, or randomly attached to one or more side chains of the polypeptide. The use of PEG for improving the therapeutic capacity for specific binding agents, e.g. peptibodies, and for humanized antibodies in particular, is described in U.S. Pat. No. 6,133,426 to Gonzales et al., issued Oct. 17, 2000.

Various means for attaching chemical moieties useful as vehicles are currently available, see, e.g., Patent Cooperation Treaty (“PCT”) International Publication No. WO 96/11953, entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water soluble polymers to the N-terminus of proteins.

A preferred polymer vehicle is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton (“kD”) to about 100 kD, more preferably from about 5 kDa to about 50 kDa, most preferably from about 5 kD to about 20 kD. The PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, maleimide, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, thiol or ester group).

A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis (see, for example, FIGS. 5 and 6 and the accompanying text herein). The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.

Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention.

An additional vehicle may also be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “vehicle” in this invention. Such vehicles should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization).

Linkers

Any “linker” group is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. The linker is preferably made up of amino acids linked together by peptide bonds. Thus, in preferred embodiments, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly)4, (Gly)5), poly(Gly-Ala), and polyalanines.

Other specific examples of linkers are:

(Gly)3Lys(Gly)4 (SEQ ID NO: 595);

(Gly)3AsnGlySer(Gly)2 (SEQ ID NO: 596);

(Gly)3Cys(Gly)4 (SEQ ID NO: 597); and

GlyProAsnGlyGly (SEQ ID NO: 598).
To explain the above nomenclature, for example, (Gly)3Lys(Gly)4 means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are also preferred. The linkers shown here are exemplary; linkers within the scope of this invention may be much longer and may include other residues.

Non-peptide linkers are also possible. For example, alkyl linkers such as —NH—(CH2)s—C(O)—, wherein s=2-20 could be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

VI

wherein n is such that the linker has a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD. The peptide linkers may be altered to form derivatives in the same manner as described above.

Derivatives

The invention also provides “derivatives” that include molecules bearing modifications other than, or in addition to, insertions, deletions, or substitutions of amino acid residues. Preferably, the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties. Derivatives of the invention may be prepared to increase circulating half-life of a molecule; to improve targeting capacity for the molecule to desired cells, tissues, or organs; to improve the solubility or absorption of a molecule; or to eliminate or attenuate any undesirable side-effect of a molecule.

Exemplary derivatives include compounds in which:

1. The compound or some portion thereof is cyclic. For example, the peptide portion may be modified to contain two or more Cys residues (e.g., in the linker), which could cyclize by disulfide bond formation.

For citations to references on preparation of cyclized derivatives, see Table 2.

2. The compound is cross-linked or is rendered capable of cross-linking between molecules. For example, the peptide portion may be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound may also be cross-linked through its C-terminus, as in the molecule shown below.

4. The N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include —NRR1 (other than —NH2), —NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1, succinimide, or benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R1 are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, chloro, and bromo.

5. The free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. For example, one may use methods described in the art to add (NH—CH2—CH2—NH2)2 to compounds of this invention. Likewise, one may use methods described in the art to add —NH2 to compounds of this invention. Exemplary C-terminal derivative groups include, for example, —C(O)R2 wherein R2 is lower alkoxy or —NR3R4 wherein R3 and R4 are independently hydrogen or C1-C8 alkyl (preferably C1-C4 alkyl).

7. One or more individual amino acid residues is modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R′—N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman & Co., San Francisco), pp. 79-86 (1983).

Such derivatized moieties preferably improve one or more characteristics including anti-angiogenic activity, solubility, absorption, biological half life, and the like of the compounds. Alternatively, derivatized moieties may result in compounds that have the same, or essentially the same, characteristics and/or properties of the compound that is not derivatized. The moieties may alternatively eliminate or attenuate any undesirable side effect of the compounds and the like.

Compounds of the present invention may be changed at the DNA level, as well. The DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes.

Isotope- and toxin-conjugated derivatives. Another set of useful derivatives are the above-described molecules conjugated to toxins, tracers, or radioisotopes. Such conjugation is especially useful for molecules comprising peptide sequences that bind to tumor cells or pathogens. Such molecules may be used as therapeutic agents or as an aid to surgery (e.g., radioimmunoguided surgery or RIGS) or as diagnostic agents (e.g., radioimmunodiagnostics or RID).

As therapeutic agents, these conjugated derivatives possess a number of advantages. They facilitate use of toxins and radioisotopes that would be toxic if administered without the specific binding provided by the peptide sequence. They also can reduce the side-effects that attend the use of radiation and chemotherapy by facilitating lower effective doses of the conjugation partner.

Useful conjugation partners include:

radioisotopes, such as 90Yttrium, 131Iodine, 225Actinium, and 213Bismuth;

ricin A toxin, microbially derived toxins such as Pseudomonas endotoxin (e.g., PE38, PE40), and the like;

partner molecules in capture systems (see below);

biotin, streptavidin (useful as either partner molecules in capture systems or as tracers, especially for diagnostic use); and

cytotoxic agents (e.g., doxorubicin).

One useful adaptation of these conjugated derivatives is use in a capture system. In such a system, the molecule of the present invention would comprise a benign capture molecule. This capture molecule would be able to specifically bind to a separate effector molecule comprising, for example, a toxin or radioisotope. Both the vehicle-conjugated molecule and the effector molecule would be administered to the patient. In such a system, the effector molecule would have a short half-life except when bound to the vehicle-conjugated capture molecule, thus minimizing any toxic side-effects. The vehicle-conjugated molecule would have a relatively long half-life but would be benign and non-toxic. The specific binding portions of both molecules can be part of a known specific binding pair (e.g., biotin, streptavidin) or can result from peptide generation methods such as those described herein.

Such conjugated derivatives may be prepared by methods known in the art. In the case of protein effector molecules (e.g., Pseudomonas endotoxin), such molecules can be expressed as fusion proteins from correlative DNA constructs. Radioisotope conjugated derivatives may be prepared, for example, as described for the BEXA antibody (Coulter). Derivatives comprising cytotoxic agents or microbial toxins may be prepared, for example, as described for the BR96 antibody (Bristol-Myers Squibb). Molecules employed in capture systems may be prepared, for example, as described by the patents, patent applications, and publications from NeoRx. Molecules employed for RIGS and RID may be prepared, for example, by the patents, patent applications, and publications from NeoProbe.

A process for preparing conjugation derivatives is also contemplated. Tumor cells, for example, exhibit epitopes not found on their normal counterparts. Such epitopes include, for example, different post-translational modifications resulting from their rapid proliferation.

Thus, one aspect of this invention is a process comprising:

a) selecting at least one randomized peptide that specifically binds to a target epitope; and

b) preparing a pharmacologic agent comprising (i) at least one vehicle (Fc domain preferred), (ii) at least one amino acid sequence of the selected peptide or peptides, and (iii) an effector molecule.
The target epitope is preferably a tumor-specific epitope or an epitope specific to a pathogenic organism. The effector molecule may be any of the above-noted conjugation partners and is preferably a radioisotope.

Variants

Variants are also included within the scope of the present invention. Included within variants are insertional, deletional, and substitutional variants. It is understood that a particular molecule of the present invention may contain one, two or all three types of variants. Insertional and substitutional variants may contain natural amino acids, unconventional amino acids (as set forth below), or both.

In one example, insertional variants are provided wherein one or more amino acid residues, either naturally occurring or unconventional amino acids, supplement a peptide or a peptibody amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the peptibody amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels. Insertional variants include peptides and peptibodies wherein one or more amino acid residues are added to the peptide or peptibody amino acid sequence, or fragment thereof.

Variants of the invention also include mature peptides and peptibodies wherein leader or signal sequences are removed, and the resulting proteins having additional amino terminal residues, which amino acids may be natural or non-natural. Molecules of this invention (such as peptibodies) with an additional methionyl residue at amino acid position −1 (Met−1-peptibody) are contemplated, as are specific binding agents with additional methionine and lysine residues at positions −2 and −1 (Met−2-Lys−1-). Variants having additional Met, Met-Lys, Lys residues (or one or more basic residues, in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.

The invention also embraces variants having additional amino acid residues that arise from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at amino acid position −1 after cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated, including those wherein poly-histidine tags are incorporated into the amino acid sequence, generally at the carboxy and/or amino terminus of the sequence.

Insertional variants also include fusion proteins wherein the amino and/or carboxy termini of the peptide or peptibody is fused to another polypeptide, a fragment thereof or amino acids which are not generally recognized to be part of any specific protein sequence. Examples of such fusion proteins are immunogenic polypeptides, proteins with long circulating half lives, such as immunoglobulin constant regions, marker proteins, proteins or polypeptides that facilitate purification of the desired peptide or peptibody, and polypeptide sequences that promote formation of multimeric proteins (such as leucine zipper motifs that are useful in dimer formation/stability).

This type of insertional variant generally has all or a substantial portion of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide. For example, fusion proteins typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion protein includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.

There are various commercially available fusion protein expression systems that may be used in the present invention. Particularly useful systems include but are not limited to the glutathione-S-transferase (GST) system (Pharmacia), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6xHis system (Qiagen, Chatsworth, Calif.). These systems are capable of producing recombinant peptides and/or peptibodies bearing only a small number of additional amino acids, which are unlikely to significantly affect the activity of the peptide or peptibody. For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of a polypeptide to its native conformation. Another N-terminal fusion that is contemplated to be useful is the fusion of a Met-Lys dipeptide at the N-terminal region of the protein or peptides. Such a fusion may produce beneficial increases in protein expression or activity.

Other fusion systems produce polypeptide hybrids where it is desirable to excise the fusion partner from the desired peptide or peptibody. In one embodiment, the fusion partner is linked to the recombinant peptibody by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

The invention also provides fusion polypeptides which comprises all or part of a peptibody or peptide of the present invention, in combination with truncated tissue factor (tTF). tTF is a vascular targeting agent consisting of a truncated form of a human coagulation-inducing protein that acts as a tumor blood vessel clotting agent, as described U.S. Pat. Nos. 5,877,289; 6,004,555; 6,132,729; 6,132,730; 6,156,321; and European Patent No. EP 0988056. The fusion of tTF to the anti-Ang-2 peptibody or peptide, or fragments thereof facilitates the delivery of anti-Ang-2 to target cells.

In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a peptide or peptibody are removed. Deletions can be effected at one or both termini of the peptibody, or from removal of one or more residues within the peptibody amino acid sequence. Deletion variants necessarily include all fragments of a peptide or peptibody.

In still another aspect, the invention provides substitution variants of peptides and peptibodies of the invention. Substitution variants include those peptides and peptibodies wherein one or more amino acid residues are removed and replaced with one or more alternative amino acids, which amino acids may be naturally occurring or non-naturally occurring. Substitutional variants generate peptides or peptibodies that are “similar” to the original peptide or peptibody, in that the two molecules have a certain percentage of amino acids that are identical. Substitution variants include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, amino acids within a peptide or peptibody, wherein the number of substitutions may be up to ten percent or more, of the amino acids of the peptide or peptibody. In one aspect, the substitutions are conservative in nature, however, the invention embraces substitutions that are also non-conservative and also includes unconventional amino acids.

Preferred methods to determine the relatedness or percent identity of two peptides or polypeptides, or a polypeptide and a peptide, are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res., 12:387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis., BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra (1990)). The well-known Smith Waterman algorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences may result in the matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, in certain embodiments, the selected alignment method (GAP program) will result in an alignment that spans at least ten percent of the full length of the target polypeptide being compared, i.e. at least 40 contiguous amino acids where sequences of at least 400 amino acids are being compared, 30 contiguous amino acids where sequences of at least 300 to about 400 amino acids are being compared, at least 20 contiguous amino acids where sequences of 200 to about 300 amino acids are being compared, and at least 10 contiguous amino acids where sequences of about 100 to 200 amino acids are being compared.

For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.), two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the “matched span”, as determined by the algorithm). In certain embodiments, a gap opening penalty (which is typically calculated as 3× the average diagonal; the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see Dayhoff et al., Atlas of Protein Sequence and Structure, 5(3)(1978) for the PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also used by the algorithm.

In certain embodiments, the parameters for a polypeptide sequence comparison include the following:

Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);

Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992);

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program may be useful with the above parameters. In certain embodiments, the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

In certain embodiments, the parameters for polynucleotide molecule sequence (as opposed to an amino acid sequence) comparisons include the following:

Algorithm: Needleman et al., supra (1970);

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

The GAP program may also be useful with the above parameters. The aforementioned parameters are the default parameters for polynucleotide molecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, thresholds of similarity, etc. may be used, including those set forth in the Program Manual, Wisconsin Package, Version 9, September, 1997. The particular choices to be made will be apparent to those of skill in the art and will depend on the specific comparison to be made, such as DNA-to-DNA, protein-to-protein, protein-to-DNA; and additionally, whether the comparison is between given pairs of sequences (in which case GAP or BestFit are generally preferred) or between one sequence and a large database of sequences (in which case FASTA or BLASTA are preferred).

The amino acids may have either L or D stereochemistry (except for Gly, which is neither L nor D) and the polypeptides and compositions of the present invention may comprise a combination of stereochemistries. However, the L stereochemistry is preferred. The invention also provides reverse molecules wherein the amino terminal to carboxy terminal sequence of the amino acids is reversed. For example, the reverse of a molecule having the normal sequence X1-X2-X3 would be X3-X2-X1. The invention also provides retro-reverse molecules wherein, as above, the amino terminal to carboxy terminal sequence of amino acids is reversed and residues that are normally “L” enantiomers are altered to the “D” stereoisomer form.

Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5′ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5′ direction. The direction of 5′ to 3′ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5′ to the 5′ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA and which are 3′ to the 3′ end of the RNA transcript are referred to as “downstream sequences”.

It will be appreciated that amino acid residues can be divided into classes based on their common side chain properties:

Conservative amino acid substitutions may encompass unconventional amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, without limitation, peptidomimetics and other reversed or inverted forms of amino acid moieties. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al. J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those which are within ±1 are included, and in certain embodiments, those within ±0.5 are included.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional peptibody or peptide thereby created is intended for use in immunological embodiments, as in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5) and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those which are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”

Exemplary amino acid substitutions are set forth in Table 10 below.

TABLE 10

Amino Acid Substitutions

Original

Preferred

Residues

Exemplary Substitutions

Substitutions

Ala

Val, Leu, Ile

Val

Arg

Lys, Gln, Asn

Lys

Asn

Gln, Glu, Asp

Gln

Asp

Glu, Gln, Asp

Glu

Cys

Ser, Ala

Ser

Gln

Asn, Glu, Asp

Asn

Glu

Asp, Gln, Asn

Asp

Gly

Pro, Ala

Ala

His

Asn, Gln, Lys, Arg

Arg

Ile

Leu, Val, Met, Ala, Phe,

Leu

Norleucine

Leu

Norleucine, Ile, Val, Met,

Ile

Ala, Phe

Lys

Arg, 1,4 Diamino-butyric

Arg

Acid, Gln, Asn

Met

Leu, Phe, Ile

Leu

Phe

Leu, Val, Ile, Ala, Tyr

Leu

Pro

Ala

Gly

Ser

Thr, Ala, Cys

Thr

Thr

Ser

Ser

Trp

Tyr, Phe

Tyr

Tyr

Trp, Phe, Thr, Ser

Phe

Val

Ile, Met, Leu, Phe, Ala,

Leu

Norleucine

A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar peptides or polypeptides. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou et al., Biophys. T., 26:367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate.

In certain embodiments, peptibody variants include glycosylation variants wherein one or more glycosylation sites, such as a N-linked glycosylation site, has been added to the peptibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution or addition of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created.

Compounds of the present invention may be changed at the DNA level, as well. The DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes. Thus, all modifications, substitution, derivitizations, etc. discussed herein apply equally to all aspects of the present invention, including but not limited to peptides, peptide dimers and multimers, linkers, and vehicles.

Affinity Maturation

One embodiment of the present invention includes “affinity matured” peptides and peptibodies. This procedure contemplates increasing the affinity or the bio-activity of the peptides and peptibodies of the present invention using phage display or other selection technologies. Based on a consensus sequence (which is generated for a collection of related peptides), directed secondary phage display libraries can be generated in which the “core” amino acids (determined from the consensus sequence) are held constant or are biased in frequency of occurrence. Alternatively, an individual peptide sequence can be used to generate a biased, directed phage display library. Panning of such libraries can yield peptides (which can be converted to peptibodies) with enhanced binding to the target or with enhanced bio-activity.

Non-Peptide Analogs/Protein Mimetics

Furthermore, non-peptide analogs of peptides that provide a stabilized structure or lessened biodegradation, are also contemplated. Peptide mimetic analogs can be prepared based on a selected inhibitory peptide by replacement of one or more residues by nonpeptide moieties. Preferably, the nonpeptide moieties permit the peptide to retain its natural confirmation, or stabilize a preferred, e.g., bioactive, confirmation which retains the ability to recognize and bind Ang-2. In one aspect, the resulting analog/mimetic exhibits increased binding affinity for Ang-2. One example of methods for preparation of nonpeptide mimetic analogs from peptides is described in Nachman et al., Regul. Pept. 57:359-370 (1995). If desired, the peptides of the invention can be modified, for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the peptides of the invention. The peptibodies also can be modified to create peptide derivatives by forming covalent or noncovalent complexes with other moieties. Covalently-bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptibodies, or at the N- or C-terminus.

In particular, it is anticipated that the peptides can be conjugated to a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a colorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). The invention accordingly provides a molecule comprising a peptibody molecule, wherein the molecule preferably further comprises a reporter group selected from the group consisting of a radiolabel, a fluorescent label, an enzyme, a substrate, a solid matrix, and a carrier. Such labels are well known to those of skill in the art, e.g., biotin labels are particularly contemplated. The use of such labels is well known to those of skill in the art and is described in, e.g. U.S. Pat. Nos. 3,817,837; 3,850,752; 3,996,345; and U.S. Pat. No. 4,277,437. Other labels that will be useful include but are not limited to radioactive labels, fluorescent labels and chemiluminescent labels. U.S. Patents concerning use of such labels include, for example, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; and U.S. Pat. No. 3,996,345. Any of the peptibodies of the present invention may comprise one, two, or more of any of these labels.

Methods of Making

The compounds of this invention largely may be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used.

The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.

The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art.

Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art.

The compounds may also be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides.

Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques.

Uses of the Compounds

In general. The compounds of this invention have pharmacologic activity resulting from their ability to bind to proteins of interest as agonists, mimetics or antagonists of the native ligands of such proteins of interest. The utility of specific compounds is shown in Table 2. The activity of these compounds can be measured by assays known in the art. For the TPO-mimetic and EPO-mimetic compounds, in vivo assays are further described in the Examples section herein.

In addition to therapeutic uses, the compounds of the present invention are useful in diagnosing diseases characterized by dysfunction of their associated protein of interest. In one embodiment, a method of detecting in a biological sample a protein of interest (e.g., a receptor) that is capable of being activated comprising the steps of: (a) contacting the sample with a compound of this invention; and (b) detecting activation of the protein of interest by the compound. The biological samples include tissue specimens, intact cells, or extracts thereof. The compounds of this invention may be used as part of a diagnostic kit to detect the presence of their associated proteins of interest in a biological sample. Such kits employ the compounds of the invention having an attached label to allow for detection. The compounds are useful for identifying normal or abnormal proteins of interest. For the EPO-mimetic compounds, for example, presence of abnormal protein of interest in a biological sample may be indicative of such disorders as Diamond Blackfan anemia, where it is believed that the EPO receptor is dysfunctional.

Therapeutic uses of EPO-mimetic Molecules The EPO-mimetic compounds of the invention are useful for treating disorders characterized by low red blood cell levels. Included in the invention are methods of modulating the endogenous activity of an EPO receptor in a mammal, preferably methods of increasing the activity of an EPO receptor. In general, any condition treatable by erythropoietin, such as anemia, may also be treated by the EPO-mimetic compounds of the invention. These compounds are administered by an amount and route of delivery that is appropriate for the nature and severity of the condition being treated and may be ascertained by one skilled in the art. Preferably, administration is by injection, either subcutaneous, intramuscular, or intravenous.

Therapeutic uses of TPO-mimetic Compounds

For the TPO-mimetic compounds, one can utilize such standard assays as those described in WO95/26746 entitled “Compositions and Methods for Stimulating Megakaryocyte Growth and Differentiation”. In vivo assays also appear in the Examples hereinafter.

The conditions to be treated are generally those that involve an existing megakaryocyte/platelet deficiency or an expected megakaryocyte/platelet deficiency (e.g., because of planned surgery or platelet donation). Such conditions will usually be the result of a deficiency (temporary or permanent) of active Mpl ligand in vivo. The generic term for platelet deficiency is thrombocytopenia, and hence the methods and compositions of the present invention are generally available for treating thrombocytopenia in patients in need thereof.

Thrombocytopenia (platelet deficiencies) may be present for various reasons, including chemotherapy and other therapy with a variety of drugs, radiation therapy, surgery, accidental blood loss, and other specific disease conditions. Exemplary specific disease conditions that involve thrombocytopenia and may be treated in accordance with this invention are: aplastic anemia, idiopathic thrombocytopenia, metastatic tumors which result in thrombocytopenia, systemic lupus erythematosus, splenomegaly, Fanconi's syndrome, vitamin B12 deficiency, folic acid deficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria. Also, certain treatments for AIDS result in thrombocytopenia (e.g., AZT). Certain wound healing disorders might also benefit from an increase in platelet numbers.

With regard to anticipated platelet deficiencies, e.g., due to future surgery, a compound of the present invention could be administered several days to several hours prior to the need for platelets. With regard to acute situations, e.g., accidental and massive blood loss, a compound of this invention could be administered along with blood or purified platelets.

The TPO-mimetic compounds of this invention may also be useful in stimulating certain cell types other than megakaryocytes if such cells are found to express Mpl receptor. Conditions associated with such cells that express the Mpl receptor, which are responsive to stimulation by the Mpl ligand, are also within the scope of this invention.

The TPO-mimetic compounds of this invention may be used in any situation in which production of platelets or platelet precursor cells is desired, or in which stimulation of the c-Mpl receptor is desired. Thus, for example, the compounds of this invention may be used to treat any condition in a mammal wherein there is a need of platelets, megakaryocytes, and the like. Such conditions are described in detail in the following exemplary sources:

The TPO-mimetic compounds of this invention may also be useful in maintaining the viability or storage life of platelets and/or megakaryocytes and related cells. Accordingly, it could be useful to include an effective amount of one or more such compounds in a composition containing such cells.

Therapeutic uses of Ang-2 Binding Molecules

Agents that modulate Ang-2 binding activity, or other cellular activity, may be used in combination with other therapeutic agents to enhance their therapeutic effects or decrease potential side effects.

In one aspect, the present invention provides reagents and methods useful for treating diseases and conditions characterized by undesirable or aberrant levels of Ang-2 activity in a cell. These diseases include cancers, and other hyperproliferative conditions, such as hyperplasia, psoriasis, contact dermatitis, immunological disorders, and infertility.

The present invention also provides methods of treating cancer in an animal, including humans, comprising administering to the animal an effective amount of a specific binding agent, such as a peptibody, that inhibits or decreases Ang-2 activity. The invention is further directed to methods of inhibiting cancer cell growth, including processes of cellular proliferation, invasiveness, and metastasis in biological systems. Methods include use of a compound of the invention as an inhibitor of cancer cell growth. Preferably, the methods are employed to inhibit or reduce cancer cell growth, invasiveness, metastasis, or tumor incidence in living animals, such as mammals. Methods of the invention are also readily adaptable for use in assay systems, e.g., assaying cancer cell growth and properties thereof, as well as identifying compounds that affect cancer cell growth.

The cancers treatable by methods of the present invention preferably occur in mammals. Mammals include, for example, humans and other primates, as well as pet or companion animals such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep, and cattle.

Tumors or neoplasms include growths of tissue cells in which the multiplication of the cells is uncontrolled and progressive. Some such growths are benign, but others are termed malignant and may lead to death of the organism. Malignant neoplasms or cancers are distinguished from benign growths in that, in addition to exhibiting aggressive cellular proliferation, they may invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized in that they show a greater loss of differentiation (greater dedifferentiation), and of their organization relative to one another and their surrounding tissues. This property is also called “anaplasia.”

Neoplasms treatable by the present invention also include solid tumors, i.e., carcinomas and sarcomas. Carcinomas include those malignant neoplasms derived from epithelial cells that infiltrate (invade) the surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular tissue, or which form recognizable glandular structures. Another broad category or cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance like embryonic connective tissue. The invention also enables treatment of cancers of the myeloid or lymphoid systems, including leukemias, lymphomas and other cancers that typically do not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems.

The myostatin binding agents of the present invention bind to myostatin and block or inhibit myostatin signaling within targeted cells. The present invention provides methods and reagents for reducing the amount or activity of myostatin in an animal by administering an effective dosage of one or more myostatin binding agents to the animal. In one aspect, the present invention provides methods and reagents for treating myostatin-related disorders in an animal comprising administering an effective dosage of one or more binding agents to the animal. These myostatin-related disorders include but are not limited to various forms of muscle wasting, as well as metabolic disorders such as diabetes and related disorders, and bone degenerative diseases such as osteoporosis.

As shown in the Example 8 of U.S. Ser. No. 10/742,379, exemplary peptibodies of the present invention dramatically increases lean muscle mass in the CD1 nu/nu mouse model. This in vivo activity correlates to the in vitro binding and inhibitory activity described below for the same peptibodies.

Additional muscle wasting disorders arise from chronic disease such as amyotrophic lateral sclerosis, congestive obstructive pulmonary disease, cancer, AIDS, renal failure, and rheumatoid arthritis. For example, cachexia or muscle wasting and loss of body weight was induced in athymic nude mice by a systemically administered myostatin (Zimmers et al., supra). In another example, serum and intramuscular concentrations of myostatin-immunoreactive protein was found to be increased in men exhibiting AIDS-related muscle wasting and was inversely related to fat-free mass (Gonzalez-Cadavid et al. (1998), PNAS USA 95: 14938-14943). Additional conditions resulting in muscle wasting may arise from inactivity due to disability such as confinement in a wheelchair, prolonged bedrest due to stroke, illness, bone fracture or trauma, and muscular atrophy in a microgravity environment (space flight). For example, plasma myostatin immunoreactive protein was found to increase after prolonged bedrest (Zachwieja et al. J Gravit Physiol. 6(2):11(1999). It was also found that the muscles of rats exposed to a microgravity environment during a space shuttle flight expressed an increased amount of myostatin compared with the muscles of rats which were not exposed (Lalani et al. (2000), J. Endocrin. 167 (3):417-28).

In addition, age-related increases in fat to muscle ratios, and age-related muscular atrophy appear to be related to myostatin. For example, the average serum myostatin-immunoreactive protein increased with age in groups of young (19-35 yr old), middle-aged (36-75 yr old), and elderly (76-92 yr old) men and women, while the average muscle mass and fat-free mass declined with age in these groups (Yarasheski et al. J Nutr Aging 6(5):343-8 (2002)). It has also been shown that myostatin gene knockout in mice increased myogenesis and decreased adipogenesis (Lin et al. (2002), Biochem Biophys Res Commun 291(3):701-6, resulting in adults with increased muscle mass and decreased fat accumulation and leptin secretion. Exemplary molecules improve the lean muscle mass to fat ratio in aged mdx mice as shown below.

In addition, myostatin has now been found to be expressed at low levels in heart muscle and expression is upregulated after cardiomyocytes after infarct (Sharma et al. (1999), J Cell Physiol. 180 (1):1-9). Therefore, reducing myostatin levels in the heart muscle may improve recovery of heart muscle after infarct.

Myostatin also appears to influence metabolic disorders including type 2 diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, and obesity. For example, lack of myostatin has been shown to improve the obese and diabetic phenotypes of two mouse models (Yen et al. supra). In addition, increasing muscle mass by reducing myostatin levels may improve bone strength and reduce osteoporosis and other degenerative bone diseases. It has been found, for example, that myostatin-deficient mice showed increased mineral content and density of the mouse humerus and increased mineral content of both trabecular and cortical bone at the regions where the muscles attach, as well as increased muscle mass (Hamrick et al. (2002), Calcif Tissue Int 71(1): 63-8). In the present invention, an exemplary peptibody increases the lean muscle mass to fat ratio in mdx mouse models as shown below.

The present invention also provides methods and reagents for increasing muscle mass in food animals by administering an effective dosage of the myostatin binding agent to the animal. Since the mature C-terminal myostatin polypeptide is identical in all species tested, myostatin binding agents would be expected to be effective for increasing muscle mass and reducing fat in any agriculturally important species including cattle, chicken, turkeys, and pigs.

The myostatin-binding molecules of the present invention may be used alone or in combination with other therapeutic agents to enhance their therapeutic effects or decrease potential side effects. The molecules of the present invention possess one or more desirable but unexpected combination of properties to improve the therapeutic value of the agents. These properties include increased activity, increased solubility, reduced degradation, increased half-life, reduced toxicity, and reduced immunogenicity. Thus the molecules of the present invention are useful for extended treatment regimes. In addition, the properties of hydrophilicity and hydrophobicity of the compounds of the invention are well balanced, thereby enhancing their utility for both in vitro and especially in vivo uses. Specifically, compounds of the invention have an appropriate degree of solubility in aqueous media that permits absorption and bioavailability in the body, while also having a degree of solubility in lipids that permits the compounds to traverse the cell membrane to a putative site of action, such as a particular muscle mass.

The myostatin-binding molecules of the present invention are useful for treating a “subject” or any animal, including humans, when administered in an effective dosages in a suitable composition.

In addition, the mystatin-binding molecules of the present invention are useful for detecting and quantitating myostatin in a number of assays. These assays are described in detail in U.S. Ser. No. 10/742,379.

In general, the myostatin-binding molecules of the present invention are useful as capture agents to bind and immobilize myostatin in a variety of assays, similar to those described, for example, in Asai, ed., Methods in Cell Biology 37, Antibodies in Cell Biology, Academic Press, Inc., New York (1993). The myostatin-binding molecule may be labeled in some manner or may react with a third molecule such as an anti-binding molecule antibody which is labeled to enable myostatin to be detected and quantitated. For example, a myostatin-binding molecule or a third molecule can be modified with a detectable moiety, such as biotin, which can then be bound by a fourth molecule, such as enzyme-labeled streptavidin, or other proteins. (Akerstrom (1985), J Immunol 135:2589; Chaubert (1997), Mod Pathol 10:585).

Throughout any particular assay, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures.

Therapeutic uses of BAFF-binding molecules. BAFF-binding molecules of this invention may be particularly useful in treatment of B-cell mediated autoimmune diseases. In particular, they may be useful in treating, preventing, ameliorating, diagnosing or prognosing lupus, including systemic lupus erythematosus (SLE), and lupus-associated diseases and conditions. Other preferred indications include B-cell mediated cancers, including B-cell lymphoma.

The compounds of this invention can also be used to treat inflammatory conditions of the joints. Inflammatory conditions of a joint are chronic joint diseases that afflict and disable, to varying degrees, millions of people worldwide. Rheumatoid arthritis is a disease of articular joints in which the cartilage and bone are slowly eroded away by a proliferative, invasive connective tissue called pannus, which is derived from the synovial membrane. The disease may involve peri-articular structures such as bursae, tendon sheaths and tendons as well as extra-articular tissues such as the subcutis, cardiovascular system, lungs, spleen, lymph nodes, skeletal muscles, nervous system (central and peripheral) and eyes (Silberberg (1985), Anderson's Pathology, Kissane (ed.), II:1828). Osteoarthritis is a common joint disease characterized by degenerative changes in articular cartilage and reactive proliferation of bone and cartilage around the joint. Osteoarthritis is a cell-mediated active process that may result from the inappropriate response of chondrocytes to catabolic and anabolic stimuli. Changes in some matrix molecules of articular cartilage reportedly occur in early osteoarthritis (Thonar et al. (1993), Rheumatic disease clinics of North America, Moskowitz (ed.), 19:635-657 and Shinmei et al. (1992), Arthritis Rheum., 35:1304-1308). TALL-1, TALL-1R and modulators thereof are believed to be useful in the treatment of these and related conditions.

Combination Therapy. The therapeutic methods, compositions and compounds of the present invention may also be employed, alone or in combination with other cytokines, soluble Mpl receptor, hematopoietic factors, interleukins, growth factors or antibodies in the treatment of disease states characterized by other symptoms as well as platelet deficiencies. It is anticipated that the inventive compound will prove useful in treating some forms of thrombocytopenia in combination with general stimulators of hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocytic stimulatory factors, i.e., meg-CSF, stem cell factor (SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or other molecules with megakaryocyte stimulating activity may also be employed with Mpl ligand. Additional exemplary cytokines or hematopoietic factors for such co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, or IFN-gamma. It may further be useful to administer, either simultaneously or sequentially, an effective amount of a soluble mammalian Mpl receptor, which appears to have an effect of causing megakaryocytes to fragment into platelets once the megakaryocytes have reached mature form. Thus, administration of an inventive compound (to enhance the number of mature megakaryocytes) followed by administration of the soluble Mpl receptor (to inactivate the ligand and allow the mature megakaryocytes to produce platelets) is expected to be a particularly effective means of stimulating platelet production. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by conventional methods.

In cases where the inventive compounds are added to compositions of platelets and/or megakaryocytes and related cells, the amount to be included will generally be ascertained experimentally by techniques and assays known in the art. An exemplary range of amounts is 0.1 μg-1 mg inventive compound per 106 cells.

Pharmaceutical Compositions

In General

The present invention also provides methods of using pharmaceutical compositions of the inventive compounds. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal, transdermal or other forms of administration. In general, the invention encompasses pharmaceutical compositions comprising effective amounts of a compound of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations.

Oral Dosage Forms

Contemplated for use herein are oral solid dosage forms, which are described generally in Chapter 89 of Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Chapter 10 of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, herein incorporated by reference. In general, the formulation will include the inventive compound, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the above inventive compounds. If necessary, the compounds may be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached vehicles in this invention may also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp 367-83; Newmark, et al. (1982), J. Appl. Biochem. 4:185-9. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties.

For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oral drug delivery composition and methods”.

The compounds of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.

Additives may also be included in the formulation to enhance uptake of the compound. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The compound of this invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compounds of this invention is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.

The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, for most effective delivery to the distal lung.

Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.

Other Delivery Forms

Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated.

Buccal delivery of the inventive compound is also contemplated. Buccal delivery formulations are known in the art for use with peptides.

Dosages

The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.

Specific Preferred Embodiments

The inventors have determined preferred peptide sequences for molecules having many different kinds of activity. The inventors have further determined preferred structures of these preferred peptides combined with preferred linkers and vehicles. Preferred structures for these preferred peptides listed in Table 11 below. Linker sequences are shown in bold. Active peptide sequences are shown in bold and areunderlined.

TABLE 11

Preferred embodiments

SEQ

ID

Sequence/structure

NO:

Activity

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

616

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GIEGPTLRQW

Amp2

151

LAARAGGTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD

201

GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK*

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

648

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

Loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDEL GGGTYSCHFGPL

EMP1

151

TWVCKPQGGG TKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

(1 Gly

201

SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK*

linkers)

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

649

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GTYSCHFGPL

EMP1

151

TWVCKPQGGT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

(2Gly

201

SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK*

linkers)

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

650

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GGTYSCHFGP

EMP1

151

LTWVCKPQGG GTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

(3Gly

201

LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

linkers)

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

651

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GGGTYSCHFG

EMP1

151

PLTWVCKPQG GGGTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP

(4Gly

201

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP

linkers)

251

GK*

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

652

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GGGTYSAHFG

EMP1

151

PLTWVAKPQG GGGTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP

(Cys > Ala

201

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP

variant

251

GK*

w/4Gly

linkers)

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

653

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GQGYCDEGPT

TMP20

151

LKQWLVCLGL QHSGGTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT

(2Gly

201

TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL

linkers)

251

SPGK*

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

654

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GQGYADEGPT

TMP20

151

LKQWLVALGL QHSGGTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT

(Cys > Ala

201

TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL

variant,

251

SPGK*

w/2Gly

linkers)

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

655

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GHAEGTFTSD

GLP1

151

VSSYLEGQAAKEFIAWLVKGRGGGTKNQVS LTCLVKGFYP SDIAVEWESN

(2Gly

201

GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN

linkers)

251

HYTQKSLSLS PGK*

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

656

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GGGHAEGTFT

GLP1

151

SDVSSYLEGQAAKEFIAWLV KGRGGGGGTK NQVSLTCLVK GFYPSDIAVE

(4Gly

201

WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

linkers)

251

ALHNHYTQKS LSLSPGK*

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

657

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GQEECEWDPW

ANG2

151

TCEHMGGTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD

(2Gly

201

GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK*

linkers)

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

612

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GLADHGQCIR

Myo7

151

WPWMCPPEGW EGGTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP

(2Gly

201

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP

linkers)

251

GK*

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

658

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GGGGDWTGDM

ANG1

151

QVKFDAMMFG PRKEGGGGGT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN

(4Gly

201

NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK

linkers)

251

SLSLSPGK

1

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

659

Fc-

51

DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

loop-

101

KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELG GGGGDWTGDM

ANG1

151

QVKFDAMMFG PRKEGGGDWT GDMQVKFDAM MFGPRKEGGG GGTKNQVSLT

(2 ×

201

CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR

peptide,

251

WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

w/4Gly

linkers)

WORKING EXAMPLES

The compounds described above may be prepared as described below. These examples comprise preferred embodiments of the invention and are illustrative rather than limiting.

Example 1Preparation of Fc-loop-ang2

In this example of the invention, the disulphide-constrained peptide TN8-Con4 was inserted into the human IgG1 Fc-loop domain, defined as the sequence D137E138L139T140K141 (FIG. 2A.).

TN8-Con4 QEECEWDPWTCEHM (SEQ ID NO: 147) The peptide insertion is between Fc residues Leu139 and Thr140 and includes 2 Gly residues as linkers flanking either side of the inserted peptide (FIG. 10A). The Fc-loop TN8-Con4 construct is labeled with Amgen clone #6888. The carboxy-terminal TN8-Con4 fusion peptibody (FIG. 10B) includes a 5 Gly linker and is labeled with Amgen clone #5564.

Both clones #6888 and #5564 were transformed into E. coli by conventional methods known to those familiar in the art. Both clones were found to express at high levels and almost exclusively in the insoluble inclusion body fraction (FIG. 11). The isolated inclusion body fraction (1 g) was solubilized in 6 M guanidine-HCl, 50 mM Tris, 8 mM DTT, pH 9 (10 ml) at room temperature with mixing, for 1 hour. The denatured and reduced peptibodies were refolded from the solubilized inclusion bodies by a 1:25 (v/v) dilution into a refold buffer consisting of 2 M urea, 50 mM Tris, 4 mM cysteine, 1 mM cystamine, pH 8.5. The solubilized peptibodies were added drop wise to the refold buffer at 4° C. with stirring. The refold reactions were allowed to stir for 48 hours, and then aliquots were evaluated by SDS-PAGE and reverse-phase HPLC. The Fc-loop TN8-Con4 (#6888) is considerably more homogeneous in the refold reaction than the carboxy-terminal Fc TN8-Con4 peptibody (#5564) as shown by RP-HPLC (FIG. 12).

Purification of the refolded Fc-loop TN8-Con4 and carboxy-terminal Fc TN8-Con4 was achieved using a 2-column process. First a recombinant Protein-A column was equilibrated in 2 M urea, 50 mM Tris, pH 8.5 and loaded with the filtered peptibody refold reaction. The column was then washed with 2 column volumes of equilibration buffer, followed by 2 column volumes of PBS. The peptibody fraction was eluted with 50 mM NaOAc, pH3 and quickly neutralized by a 1:4 dilution into 10 mM NaOAc, 50 mM NaCl, pH 5. The diluted Protein-A eluate was again filtered and loaded to an SP Sepharose HP cation exchange column (Pharmacia) equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5. The peptibody fractions were then eluted with a linear 50-500 mM NaCl gradient, pooled and concentrated to about 2 mg/ml. The final pools of Fc-loop TN8-Con4 (#6888) and the carboxy-terminal Fc TN8-Con4 (#5564) were evaluated by RP-HPLC (FIG. 13) and SDS-PAGE (FIG. 14). Both RP-HPLC and SDS-PAGE demonstrate that improved product homogeneity is achieved with the Fc-loop TN8-Con4 (#6888) relative to the comparable carboxy-terminal fused peptibody (#5564).

Both the Fc-loop TN8-Con4 and carboxy-terminal fused TN8-Con4 peptibodies were further evaluated in an in vitro ELISA for competitive inhibition of the angiopoietin 2 receptor. In this format the peptibody competes with an angiopoietin 2 receptor-Fc fusion for binding to immobilized angiopoietin 2. Binding of the angiopoietin 2 receptor-Fc fusion is monitored by fluorescence using an enzyme-linked immunodetection method and reported as an inhibition constant at 50% inhibition (IC50). This experiment shows that the Fc-loop TN8-Con4 is fully active relative to the carboxy-terminal Fc TN8-Con4 (FIG. 15).

Stability in vivo of the Fc-loop TN8-Con4 peptibody was compared to the carboxy-terminal TN8-Con4 peptibody in mice. In this study groups of 15 mice were dosed subcutaneously with either peptibody construct at 5 mg/kg. At 4 hours after injection, 5 mice were sacrificed and serum collected. At 24 hours, another 5 mice were harvested and likewise at 48 hours. Each individual serum sample was evaluated by western blot for detectable human IgG-Fc peptibody. Since all the serum within each 5-mouse group was very similar, the groups were pooled to allow representative samples to be run together on a single gel/blot. The result of that analysis (FIG. 16) clearly shows that both the Fc-loop TN8-Con4 peptibody and the carboxy-terminal TN8-Con4 peptibody persists in the pooled mouse sera throughout the 48-hour time course with no apparent loss. This result demonstrates that the Fc-loop designed peptibodies are not destabilized in vivo by the peptide insertion.

Example 2Preparation of Fc-loop-myo7

In another embodiment of this invention, a novel, disulphide-constrained peptide TN8-19-7 (U.S. Pat App 2004-0181033-A1, which is incorporated by reference) of the sequence:

TN8-19-07 LADHGQCIRWPWMCPPEGWE (SEQ ID NO: 365) was engineered between Leu139 and Thr140 as an internal fusion in the putative Fc-loop sequence DELTK of an IgG1 Fc sequence (FIG. 2A). An additional two Gly residues were also added at each end of the TN8-19-07 peptide as flanking linkers. The final Fc-loop TN8-19-07 sequence is given in FIG. 3A and is labeled clone # 6951. Alternatively, a carboxy terminal fusion of TN8-19-07 with the same IgG1 Fc sequence was prepared to serve as a control (FIG. 3B) and labeled clone #6826. The carboxy-terminal fusion included five Gly residues between the Fc and TN8-19-07 to serve as a linker.

Both clones #6951 and #6826 were transformed into E. coli by conventional methods used by those familiar in the art, and were found to express at high levels and almost exclusively in the insoluble inclusion body fraction (FIGS. 4A and 4B). The isolated inclusion body fraction (1 g) was solubilized in 6 M guanidine-HCl, 50 mM Tris, 8 mM DTT, pH 9 (10 ml) at room temperature with mixing for 1 hour. The denatured and reduced peptibodies were refolded from the solubilized inclusion body fraction by a 1:25 (v/v) dilution into 2 M urea, 50 mM Tris, 4 mM cysteine, 1 mM cystamine, pH 8.5. The solubilized peptibodies were added drop-wise to the refold buffer at 4° C. with stirring. The refold reactions were allowed to stir for 48 hours, and then aliquots were evaluated by SDS-PAGE and reverse-phase HPLC. The Fc-loop TN8-19-07 (#6951) was found to be considerably more homogeneous by RP-HPLC (FIG. 5) in the refold reaction than the carboxy-terminal Fc-TN8-19-07 peptibody (#6826).

Purification was achieved by a 2-column process. First a recombinant Protein-A column was equilibrated in 2 M urea, 50 mM Tris, pH 8.5 and loaded with the filtered peptibody refold reaction. The column was then washed with 2 column volumes of equilibration buffer, followed by 2 column volumes of PBS. The peptibody fraction was eluted with 50 mM NaOAc, pH 3 and quickly neutralized by a 1:4 dilution into 10 mM NaOAc, 50 mM NaCl, pH 5. The diluted Protein-A eluate was again filtered and loaded to an SP Sepharose HP cation exchange column (Pharmacia) equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5. The peptibody fractions were then eluted with a linear 50-500 mM NaCl gradient, pooled and concentrated to about 2 mg/ml. The final pools of Fc-loop TN8-19-07 (#6951) and the carboxy-terminal Fc TN8-19-07 (#6826) were evaluated by RP-HPLC (FIG. 6) and SDS-PAGE (FIG. 7). Both RP-HPLC and SDS-PAGE demonstrate that improved homogeneity in the final product is achieved with the Fc-loop TN8-19-07 (#6951) relative to the comparable carboxy-terminal fused peptibody (#6826).

An in vitro cell-based assay, which measures the inhibition of myostatin signaling activity, was used to determine the bioactivity of the Fc-loop TN8-19-07 (#6951) compared to the carboxy-terminal fusion (#6826). In this assay, both constructs were titrated against 4 nM myostatin and evaluated for their ability to inhibit the myostatin signaling activity as measured by a luciferase reporter system. The relative peptibody activities are reported as the effective concentration for 50% inhibition (EC50). This experiment shows that the Fc-loop TN8-19-07 peptibody (#6951) retains full in vitro bioactivity (FIG. 8).

Stability in vivo of the Fc-loop TN8-19-07 peptibody was compared to the carboxy-terminal TN8-19-07 peptibody in mice. In this study, groups of 15 mice were dosed subcutaneously with either peptibody construct at 5 mg/kg. At 4 hours post injection 5 mice were sacrificed and serum collected. At 24 hours another 5 mice were harvested and likewise at 48 hours. Each individual serum was evaluated by western blot for detectable human IgG-Fc peptibody. Since all the serum within each 5-mouse group was very similar, the groups were pooled to allow representative samples to be run together on a single gel/blot. The result of that analysis (FIG. 9) clearly shows that the Fc-loop TN8-19-07 peptibody persists in the pooled mouse sera throughout the 48-hour time course with no apparent loss. In contrast, the concentration of the carboxy-terminal TN8-19-07 peptibody diminishes steadily through the course of the study until it is nearly undetectable at the 48-hour time point. This result suggests that the Fc-loop design approach may confer additional in vivo stability to the TN8-19-07 peptibody.

Example 3Preparation of TN8-Con4

This molecule was prepared as described above in Example 1 and in U.S. Pat. App. No. 2003/0236192 (also PCT/US04/10989), which is hereby incorporated by reference.

Example 4Preparation of Fc-ang2-tandem

This molecule was prepared as described in U.S. 2003/0236193, published Dec. 25, 2003 (also PCT/US04/10989, filed Apr. 8, 2004), which is hereby incorporated by reference.

Example 5Preparation of TN8-19-7

This molecule was prepared as described above in example 2 and in U.S. Ser. No. 10/742,379, filed Dec. 19, 2003 (also PCT/US03/40781, filed Dec. 19, 2003), which is hereby incorporated by reference.

Example 6Preparation of Fc-loop-EMP

This molecule was prepared as previously described in example 1.

Example 7Preparation of Fc-loop-Amp2

In another embodiment of this invention a linear, non-constrained peptide, AMP 2 was inserted into the human IgG1 Fc-loop domain, defined as the sequence D137E138L139T140K141 (FIG. 2A).

AMP-2: IEGPTLRQWLAARA (SEQ ID NO: 28)

The Fc insertion is between Leu139 and Thr140 and includes 2 Gly residues as linkers flanking either side of the inserted peptide (FIG. 3D). The Fc-loop AMP 2 construct is labeled as Amgen clone #6875.

The Fc-loop AMP 2 clone (#6875) was transformed into E. coli by conventional methods known to those in the art and was found to express at high levels and almost exclusively in the insoluble inclusion body fraction (FIG. 17). The isolated inclusion body fraction (1 g) was solubilized in 6 M guanidine-HCl, 50 mM Tris, 8 mM DTT, pH 9 (10 ml) at room temperature with mixing, for 1 hour. The denatured and reduced peptibody was refolded from the solubilized inclusion body fraction by a 1:25 (v/v) dilution into 2 M urea, 50 mM Tris, 4 mM cysteine, 1 mM cystamine, pH 8.5. The solubilized peptibody was added drop wise to the refold buffer at 4° C. with stirring. The refold reactions were allowed to stir for 48 hours, and then aliquots were evaluated by SDS-PAGE and reversed-phase HPLC.

Purification was achieved using a 2-column process. First a recombinant Protein-A column was equilibrated in 2 M urea, 50 mM Tris, pH 8.5 and loaded with the filtered peptibody refold reaction. The column was then washed with 2 column volumes of equilibration buffer, followed by 2 column volumes of PBS. The peptibody fraction was eluted with 50 mM NaOAc, pH3 and quickly neutralized by a 1:4 dilution into 10 mM NaOAc, 50 mM NaCl, pH 5. The diluted Protein-A eluate was again filtered and loaded to an SP Sepharose HP cation exchange column (Pharmacia) equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5. The peptibody fractions were then eluted with a linear 50-500 mM NaCl gradient, pooled and concentrated to about 2 mg/ml. The final pools of Fc-loop AMP 2 (#6875) were evaluated by SDS-PAGE (FIG. 19) and RP-HPLC (FIG. 20).

The final preparation of Fc-loop AMP 2 was tested in an in vivo mouse bioassay against a carboxy-terminal peptibody fusion of two AMP 2 sequences linked in tandem (Fc tandem AMP2). In this comparison, the Fc-tandem-AMP2 has a total valence of four AMP 2 peptides compared to the Fc-loop AMP 2 with only two peptides. The mice received a single subcutaneous injection of 50 μg/kg of either peptibody while their platelet levels were monitored over 15 days (FIG. 21). While the Fc-tandem-AMP2 induced a significant initial platelet increase, the total response was complete by day 9. In contrast, the Fc-loop AMP 2 elicited a much smaller response that peaked at day 8 and persisted for 15 days. These results suggest that the efficacious half-life of the Fc-loop AMP 2 peptibody may be much greater than the conventional carboxy-terminal fused peptibody. The difference in overall amplitude of the response may be a consequence of the greater valence of Fc-tandem-Amp2.

Example 8Preparation of Amp2

This molecule was prepared as described above in example 7, and in U.S. Pat. No. 6,660,843.

To quantitate myostatin activity and its blockade, a luciferase reporter system was developed, referred to as pMARE-Luc, which senses Myostatin/Activin signaling strength. The pMARE-luc vector was constructed by subcloning a Smad-responsive CAGA tandem repeat sequence into a basic reporter plasmid pLuc-MCS containing a minimal promoter element (TATA box). The pMARE-luc vector was stably transfected into a skeletal muscle-derived C2C12 cell line (murine).

Characterization of myostatin responses of the stable clones led to the identification of C2C12-based clonal reporter cell lines that were capable of detecting both myostatin and Activin signaling activities in 96-well format in a highly sensitive and reproducible manner.

Starting from a concentration of 100 nM, Fc-loop peptibody and proper controls were serially diluted in HTRF buffer 3-fold, 9-times across a 96-well plate. Dilutions were then mixed with the following reagents on a 96-well black, round-bottomed assay plate: Streptavidin-Europium (1.6 nM), Biotinylated human angiopoietin-2 (8.0 nM), human Tie2-Fc-APC (10 nM). Assay plate was then incubated at room temperature with shaking for 2 hours. Plate next read on a Rubystar microplate reader (BMG Labtechnologies Inc.). Results were converted to % inhibition, and IC50s were then calculated by analyzing the %inhibition values in the program GRAFIT 5.0 (IC50, 0-100% parameter).

Example 11In Vivo AMP-2 Efficacy Assay (FIG. 21)

Female BDF1 mice are injected subcutaneously with either carrier fluid (1× PBS with 0.1% BSA), 50 mcg/kg of Fc-tandem-AMP2, or 50 mcg/kg of Fc-loop-AMP2. The injection volume is 0.2 mL. Blood is collected from each mouse via a puncture of the retro orbital sinus into a heparinized capillary tube, and then transferred to microtainers containing EDTA. Complete blood counts (CBC) including differential white blood cell counts are obtained using an ADVIA120 blood analyzer calibrated for mouse blood (Bayer Corp., Tarrytown, N.Y.). Standard bleed days are 0, 3, 5, 7 and 10. Platelet counts are plotted as a function of time post-injection.

Example 12UT-7 EPO Proliferation Assay for EMP Activity (FIG. 18)

The UT-7Epo proliferation assay uses human megakaryoblastic leukemia cell line that responds to murine EPO (mEPO) and human EPO (huEPO) or other EPO like molecules for growth and survival.

Growth factors are serially diluted from 1000 ng/ml to 0.488 ng/ml in triplicate, in 100 μl of 10% FBS-Iscoves Modified Dulbelcco's medium (IMDM) across the 96 well plate. 15000 cells/well are added to the 96 well plate in 100 μl of 10% FBS IMDM. The total volume per well is 200 μl of media with 15000 cells per well. Cells and media alone are the zero control. Cells are incubated in a humidified chamber at 37° C.

Having proven the feasibility of Fc-Loop insertion-style peptibodies with the L139/T140 insertion site using several different peptides, additional loops were surveyed in the Fc crystal structure. Using Fc-domain homology modeling, twelve different potential insertion sites were selected based on solvent accessible surface exposure, steric constraints within the loop, proximity to the Fc dimer interface and juxtaposition to sites of known effector function, such as the FcRn binding interface. The Fc-Loop sites that were identified as potential insertion sites are detailed and ranked. See FIG. 2A and Table 12 below.

TABLE 12

Specific insertion sites for the human IgG1 Fc sequence

Domain

Loop

Insertion

Comments

CH2

P25-P26

Not preferred

Tight turn

CH2

D46-E53

H49/E50 - 1st

No homology H/E site

E50/D51 - 2nd

CH2

V65-A68

Not preferred

FcRn interactive

CH2

E74-T80

Y77/N78 - 1st

Low homology Y/N site

N78/S79 - 2nd

CH2

V89-E99

Not preferred

FcRn interactive

CH2—CH3

N106-P127

K107/A108 - 1st

Exposed turn

linker

N106/K107 - 2nd

CH3

D137-K141

L139/T140 - 1st

Successfully tested

E138/L139 - 2nd

L139/T140

CH3

N165-N177

E169/N170 - 1st

Avoid tight turn N165-P168.

N170/N171 - 2nd

No homology E/N site

CH3

T175-S184

S181/D182 - 1st

No homology V/L site.

V178/L179 - 2nd

S/D site poss. better

exposed

CH3

K195-V203

G201/N202 - 1st

α/β content. G/N site

N202/V203 - 2nd

exposed. N/V site low

homology

CH3

NA

Q167/P168

IgA, IgM insertion site

CH3

NA

G183/S184

IgA, IgM insertion site

Of these potential insertion sites, six were expressed using the TN8-19-7 peptide insert (Example 2) and evaluated for refolding efficiency and in vitro activity. An additional construct was added which contained an asymmetric linker system Gly4/Gly6 engineered into the original loop insertion site (L139/T140) previously described. In all, seven new Fc Loop Myostatin constructs were refolded, purified, and tested for activity.

The seven new Fc-Loop TN8-19-7 constructs that were tested included insertions in both CH2 and CH3 domains of human IgG1 Fc. Specifically, these insertions were: G201/N202 (CH3), E169/N170 (CH3), S181/D182 (CH3), H49/E50 (CH2), L139/T140 (G4-6) (CH3), Y77/N78 (CH2), and K107/A108 (CH2-CH3 linker domain). These constructs were transformed into E. coli by conventional methods known in the art, and were found to express almost exclusively in the insoluble inclusion body fraction. Interestingly, the H49/E50, L139/T140 (G4-6) and Y77/N78 constructs appeared to have the highest levels of expression. The E169/N170, S181/D182, K107/108 showed moderate expression, and the G201/202 construct showed some expression, but very little.

Those Fc-Loop TN8-19-7 constructs which expressed well in E. coli were purified by first solubilizing the isolated inclusion body fractions in 6 M Guanidine-HCl, 50 mM Tris, 8 mM DTT pH 9.0 (10 mL per 1 g inclusion body) at room temperature with mixing for 1 hour. Then, a variety of refolding conditions were evaluated for each of the denatured and reduced Fc-Loop TN8-19-7 constructs to identify optimal refolding conditions. The three G201/N202, E169/N170, and S181/D182 constructs did not refold well under any of the conditions tested. Of the remaining four Fc-Loop TN8-19-7 constructs, L139/T140 (G4-6) refolded the best, while the remaining three H49/E50, Y77/N78 and K107/A108 refolded with sufficient yield to pursue further purification.

Using optimized refold conditions, the denatured and reduced Fc-Loop TN8-19-7 constructs were refolded from the solubilized inclusion body fractions by a 1:25 (v/v) dilution into 4 M Urea, 50 mM Tris-HCl, 0.16 M Arg-HCl, 20% glycerol, 3 M Cystine, 5 mM Cystarnine pH 8.5. The solubilized peptibodies were added drop-wise to the refold buffer at 4° C. with stirring. The refold reactions were allowed to stir for 72 hours, and subsequently purified chromatographically. Final purification was achieved by a 2-column chromatographic process, as described in Example 2.

The final pools of L139/T140 (G4-6), H49/E50, Y77/N78, and K107/108 were evaluated by RP-HPLC and SDS-PAGE, as illustrated in FIGS. 23 and 24. The yields from these four constructs are tabulated in Table 13.

TABLE 13

Expression and purification yields of recoverable Fc-loop constructs

Grams IB per

mg product per

Insertion Site

Grams Paste

gram of IB

H49/E50 (CH2 domain)

0.182

2.08

L139/T140 (G4-6)

0.156

14.58

(CH3 domain)

Y77/N78 (CH2 domain)

0.162

10.26

K107/A108

0.140

0.22

Among the four analogs purified the L139/T140 (G4-6) insertion site analog was produced with the best purity and in the highest yield.

The purified Fc-loop insertion analogs were further analyzed for functional myostatin receptor binding activity using an in vitro cell based inhibition assay, as described in Example 9. The results are shown in Table 14.

TABLE 14

In vitro cell based myostatin inhibition assay results

Fc-Loop insertion analogs

IC50(nM)

H49/E50

13.94

L139/T140 (G4-6)

0.8727

Y77/N78

17.06

Fc-Loop #6951 (139/140)

0.8335

The purified Fc-loop insertion analogs were further analyzed for FcRn binding using a Biacore assay system. Sample K107/A108 was not tested due to insufficient sample remaining after analysis. The IC50 values determined using the in vitro cell based Myostatin inhibition assay, were similar for the L139/T140 (G4-6) insertion and the original Fc-Loop #6951, whereas the Fc-loop insertions at H49/E50 and Y77/N78 showed a reduced ability to inhibit myostatin (FIG. 30). The Biacore FcRn binding experiments showed that H49/E50 and Y77/N78 bound the Fc receptor comparably to the control (Fc-Loop-1×TN8-19-7, #6951) with an EC50 of about 680 nM, but L139/T140 (G4-6) had a lower and more favorable EC50 at around 220 nM.

In summary, of the six new insertion sites evaluated, three failed to refold efficiently. Among the three new insertion site analogs recovered, K107/A108 folded poorly, H49/E50 (CH2 domain) refolded marginally well, and the two remaining insertions at Y77/N78 (CH2 domain) and the original insertion site of L139/T140 (CH3 domain) with an extended, asymmetric linker folded with a higher efficiency. Interestingly, all of these novel insertion site analogs refolded with significantly lower yield than the original Fc-loop construct (#6951) with TN8-19-7 in position L139/T140 using symmetric Gly2 linkers.

When tested for retention of FcRn binding capacity, all the Fc-loop molecules appeared similar in affinity with the possible exception of the extended, asymmetric linker construct, which seemed slightly better. This was consistent with the design paradigm to minimize steric interactions between the inserted peptide and the FcRn binding interface.

While all the purified Fc-loop constructs were active by the in vitro, cell-based functional assay (Table 14), the original insertion site (L139/T140) and the extended, asymmetric linker insertion at the same site appeared to be the most potent.

This work demonstrates that multiple loop domains within the human IgG1 Fc, as identified in FIG. 23, will tolerate insertion of bioactive peptides while preserving the activity of both the peptide and Fc effector functions such as FcRn binding. Peptide insertion analogs utilizing these Fc-loop domains can vary significantly in refolding efficiency and peptide activity. Each peptide/insertion combination can be individually optimized to maximize recovery and potency.

More preferable would be peptide insertions targeting the underlined sub-domains in FIG. 23. Most preferable are the insertion site (L139/T140) and two additional loops in the CH2 domain (H49/E50 and Y77/N78).

ABBREVIATIONS

Abbreviations used throughout this specification are as defined below, unless otherwise defined in specific circumstances.

Depending on the dose or concentration of endotoxin, the effects may either be deleterious or advantageous to the host. if excessive tnf-.alpha.,il-1, il-6, and il-8 are produced, they can evoke endotoxic (or septic) shock with symptoms

Amino acid sequence of ligand binding partner protein fused to c-terminus to n-terminus of immunoglobulin and cell surface adhesion molecules and ligands, and are useful in therapeutic or diagnostic compositions and methods.